Anti-proliferative compounds, compositions, and methods of use thereof

ABSTRACT

Compounds and compositions of Formula I are described, useful as anti-proliferative agents, and in particular anti-HPV, 
     
       
         
         
             
             
         
       
     
     wherein:
         Y 1A  and Y 1B  are independently Y 1 ;   R X1  and R X2  are independently R X ;   Y 1  is ═O, —O(R X ), ═S, —N(R X ), —N(O)(R X ), —N(OR X ), —N(O)(OR X ), or —N(N(R X )(R X ));   R X  is independently R 1 , R 2 , R 4 , W 3 , or a protecting group;   R 1  is independently —H or alkyl of 1 to 18 carbon atoms;   R 2  is independently R 3  or R 4  wherein each R 4  is independently substituted with 0 to 3 R 3  groups or taken together at a carbon atom, two R 2  groups form a ring of 3 to 8 carbons and the ring may be substituted with 0 to 3 R 3  groups;   R 3  is R 3a , R 3b , R 3c  or R 3d , provided that when R 3  is bound to a heteroatom, then R 3  is R 3c  or R 3d ;   R 3a  is —H, —F, —Cl, —Br, —I, —CF 3 , —CN, N 3 , —NO 2 , or —OR 4 ;   R 3b  is ═O, —O(R 4 ), ═S, —N(R 4 ), —N(O)(R 4 ), —N(OR 4 ), —N(O)(OR 4 ), or —N(N(R 4 )(R 4 ));   R 3c  is —R 4 , —N(R 4 )(R 4 ), —SR 4 , —S(O)R 4 , —S(O) 2 R 4 , —S(O)(OR 4 ), —S(O) 2 (OR 4 ), —OC(R 3b )R 4 , —OC(R 3b )OR 4 , —OC(R 3b )(N(R 4 )(R 4 )), —SC(R 3b )R 4 , —SC(R 3b )OR 4 , —SC(R 3b )(N(R 4 )(R 4 )), —N(R 4 )C(R 3b )R 4 , —N(R 4 )C(R 3b )OR 4 , —N(R 4 )C(R 3b ) (N(R 4 )(R 4 )), W 3  or —R 5 W 3 ;   R 3d  is —C(R 3b )R 4 , —C(R 3b )OR 4 , —C(R 3b )W 3 , —C(R 3b )OW 3  or —C(R 3b )(N(R 4 )(R 4 ));   R 4  is —H, or an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbon atoms, or alkynyl of 2 to 18 carbon atoms;   R 5  is alkylene of 1 to 18 carbon atoms, alkenylene of 2 to 18 carbon atoms, or alkynylene of 2 to 18 carbon atoms;   W 3  is W 4  or W 5 ;   W 4  is R 6 , —C(R 3b )R 6 , —C(R 3b )W 5 , —SO M2 R 6 , or —SO M2 W 5 , wherein R 6  is R 4  wherein each R 4  is substituted with 0 to 3 R 3  groups;   W 5  is carbocycle or heterocycle wherein W 5  is independently substituted with 0 to 3 R 2  groups; and   M2 is 0, 1 or 2;
 
and pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds and compositions and methodsof use thereof, useful for treating viral infections, in particularhuman papillomavirus.

2. Background

Human papillomavirus (HPV) is one of the most prevalent sexuallytransmitted infections in the world. There are more than 100 differenttypes of HPV, the majority of which are harmless. However, there areabout 30 types that are spread through sexual contact. Some types of HPVcause genital warts, which appear as single or multiple bumps in thegenital areas of men and women including the vagina, cervix, vulva (areaoutside of the vagina), penis, and rectum. Although many people infectedwith HPV have no symptoms.

While most HPV subtypes result in benign lesions, certain subtypes canlead to more serious lesions. Anogenital infections arising from HPV-16and HPV-18, while less common than HPV-6 and HPV-11, are most oftenassociated with precancerous lesions in cervical and anal tissues calleddysplasias. Patients with dysplasias are often asymptomatic and may onlydiscover their lesion after screening. High-grade dysplasias, if leftuntreated, may transform into cancerous tissues. Low-grade lesions mayspontaneously regress, while others may progress to high-grade lesions.HPV-16 and HPV-18 are most often associated with dysplasias, thoughseveral other transforming HPV subtypes are also associated withdysplasias. Recent studies indicate that up to 89% of HIV positivehomosexual males may be infected with these high-risk subtypes of HPV.HIV positive patients are also more likely to be infected with multiplesubtypes of HPV at the same time, which is associated with a higher riskof dysplasia progression.

Genital warts are the most common sexually transmitted disease in theworld and are most prevalent in people 17-33 years of age. HPV-6 andHPV-11 are responsible for nearly 90% of all genital warts, but arerarely associated with neoplastic growths. According to the AmericanSocial Health Association, at least 20 million people in the US arecurrently infected with HPV, with 5.5 million new cases of sexuallytransmitted HPV infections occurring annually. Genital warts usuallyproduce painless-itchy bumps located on or near the genitalia, butwithout treatment, may progress to larger more pronouncedcauliflower-like growths. Roughly two-thirds of people who have sexualcontact with a person infected with genital warts will develop wartswithin three months of contact. Spontaneous regression of genital wartsoccurs in 10-20% of genital wart cases. However, even if a lesionregresses, recurrence of genital warts is common with 50% recurrenceafter one year. As a result of the unsightly lesions, treatment ofgenital warts is common.

Evidence over the last two decades has led to a broad acceptance thatHPV infection is necessary, though not sufficient, for the developmentof cervical cancer. The presence of HPV in cervical cancer is estimatedat 99.7%. Anal cancer is thought to have a similar association betweenHPV infection and the development of anal dysplasia and anal cancer asis the case with cervical cancer. In one study of HIV negative patientswith anal cancer, HPV infection was found in 88% of anal cancers. In theUS in 2003, 12,200 new cases of cervical cancer and 4,100cervical-cancer deaths are predicted along with 4,000 new cases of analcancer and 500 anal-cancer deaths. While the incidence of cervicalcancer has decreased in the last four decades due to widespreadscreening, the incidence of anal cancer is increasing. The increase inanal cancer incidence may be attributed in part to HIV infection sinceHIV positive patients have a higher incidence of anal cancer than thegeneral population. While anal cancer has an incidence of 0.9 cases per100,000 in the general population, anal cancer has an incidence of 35cases per 100,000 in the homosexual male population and 70-100 cases per100,000 in the HIV positive homosexual male population. In fact, due tothe high prevalence of anal dysplasia among HIV-infected patients and agrowing trend of anal cancers, the 2003 USPHA/IDSA Guidelines for theTreatment of Opportunistic Infections in HIV Positive Patients willinclude treatment guidelines for patients diagnosed with anal dysplasia.

There is no known cure for HPV. There are treatments for genital warts,though they often disappear even without treatment. The method oftreatment depends on factors such as the size and location of thegenital warts. Among the treatments used are, Imiquimod cream, 20percent podophyllin antimitotic solution, 0.5 percent podofiloxsolution, 5 percent 5-fluorouracil cream, and Trichloroacetic acid. Theuse of podophyllin or podofilox is not recommended for pregnant womenbecause they are absorbed by the skin and may cause birth defects. Theuse of 5-fluorouracil cream is also not recommended for pregnant women.Small genital warts can be physically removed by freezing (cryosurgery),burning (electrocautery) or laser treatment. Large warts that do notresponded to other treatment may have to be removed by surgery. Genitalwarts have been known to return following physical removal, in theseinstances α-interferon have been used to directly inject into thesewarts. However, α-interferon is expensive, and its use does not reducethe rate of return of the genital warts.

As such there exists an unmet need for effective HPV treatment. It hasnow been surprisingly discovered compounds that meet this need, andprovide other benefits as well.

SUMMARY OF THE INVENTION

A compound of formula I,

wherein:

Y^(1A) and Y^(1B) are independently Y¹;

R^(X1) and R^(X2) are independently R^(X);

Y¹ is ═O, —O(R^(X)), ═S, —N(R^(X)), —N(O)(R^(X)), —N(OR^(X)),—N(O)(OR^(X)), or —N(N(R^(X)) (R^(X)));

R^(X) is independently R¹, R², R⁴, W³, or a protecting group;

R¹ is independently —H or alkyl of 1 to 18 carbon atoms;

R² is independently R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is —H, —F, —Cl, —Br, —I, —CF₃, —CN, —N₃, —NO₂, or —OR⁴;

R^(3b) is ═O, —O(R⁴), ═S, —N(R⁴), —N(O)(R⁴), —N(OR⁴), —N(O)(OR⁴) or—N(N(R⁴)(R⁴));

R^(3c) is —R⁴, —N(R⁴)(R⁴), —SR⁴, —S(O)R⁴, —S(O)₂R⁴, —S(O)(OR⁴),—S(O)₂(OR⁴), —OC(R^(3b))R⁴, —OC(R^(3b))OR⁴, —OC(R^(3b))(N(R⁴)(R⁴)),—SC(R^(3b))R⁴, —SC(R^(3b))OR⁴, —SC(R^(3b))(N(R⁴)(R⁴)),—N(R⁴)C(R^(3b))R⁴, —N(R⁴)C(R^(3b))OR⁴, —N(R⁴)C(R^(3b))(N(R⁴)(R⁴)), W³ or—R⁵W³;

R^(3d) is —C(R^(3b))R⁴, —C(R^(3b))OR⁴, —C(R^(3b))W³, —C(R^(3b))OW³ or—C(R^(3b))(N(R⁴)(R⁴));

R⁴ is —H, or an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbonatoms, or alkynyl of 2 to 18 carbon atoms;

R⁵ is alkylene of 1 to 18 carbon atoms, alkenylene of 2 to 18 carbonatoms, or alkynylene of 2 to 18 carbon atoms;

W³ is W⁴ or W⁵;

W⁴ is R⁶, —C(R^(3b))R⁶, —C(R^(3b))W⁵, —SO_(M2)R⁶, or SO_(M2)W⁵, whereinR⁶ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups; and

M2 is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of the formula,

wherein:

A is

Y^(1A) and Y^(1B) are independently Y¹;

R^(X1) and R^(X2) are independently R^(X);

Y¹ is ═O, —O(R^(X)), ═S, —N(R^(X)), —N(O)(R^(X)), —N(OR^(X)),—N(O)(OR^(X)), or —N(N(R^(X))(R^(X)));

R^(X) is independently R¹, R², R⁴, W³, or a protecting group;

R¹ is independently —H or alkyl of 1 to 18 carbon atoms;

R² is independently R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is —H, —F, —Cl, —Br, —I, —CF₃, —CN, N₃, —NO₂, or —OR⁴;

R^(3b) is ═O, —O(R⁴), ═S, —N(R⁴), —N(O)(R⁴), —N(OR⁴), —N(O)(OR⁴), or—N(N(R⁴)(R⁴));

R^(3c) is —R⁴, —N(R⁴)(R⁴), —SR⁴, —S(O)R⁴, —S(O)R⁴, —S(O)(OR⁴),—S(O)₂(OR⁴), —OC(R^(3b))R⁴, —OC(R^(3b))OR⁴, —OC(R^(3b))(N(R⁴)(R⁴),—SC(R^(3b))R⁴, —SC(R^(33b))OR⁴, —SC(R^(3b))(N(R⁴)(R⁴)),—N(R⁴)C(R^(3b))R⁴, —N(R⁴)C(R^(3b))OR⁴, —N(R⁴)C(R^(3b))(N(R⁴)(R⁴)), W³ or—R⁵W³;

R^(3d) is —C(R^(3b))R⁴, —C(R^(3b))OR⁴, —C(R^(3b))W³, —C(W^(3b))W³ or—C(R^(3b))(N(R⁴)(R⁴));

R⁴ is —H, or an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbonatoms, or alkynyl of 2 to 18 carbon atoms;

R⁵ is alkylene of 1 to 18 carbon atoms, alkenylene of 2 to 18 carbonatoms, or alkynylene of 2 to 18 carbon atoms;

W³ is W⁴ or W⁵;

W⁴ is R⁶, —C(R^(3b))R⁶, —C(R^(3b))W⁵, —SO_(M2)R⁶, or —SO_(M2)W⁵, whereinR⁶ is R⁴ wherein each R⁴ is substituted with 0 to 3 R¹ groups;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups; and

M2 is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

An embodiment of the present invention provides a compound of Formula I,

wherein:

Y^(1A) and Y^(1B) are independently Y¹;

R^(X1) and R^(X2) are independently R^(X);

Y¹ is ═O, —O(R^(X)), ═S, —N(R^(X)), —N(O)(R^(X)), —N(OR^(X)),—N(O)(OR^(X)), or —N(N(R^(X))(R^(X)));

R^(X) is independently R¹, R², R⁴, W³, or a protecting group;

R¹ is independently —H or alkyl of 1 to 18 carbon atoms;

R² is independently R³ or R⁴ wherein each R⁴ is independentlysubstituted with 0 to 3 R³ groups or taken together at a carbon atom,two R² groups form a ring of 3 to 8 carbons and the ring may besubstituted with 0 to 3 R³ groups;

R³ is R^(3a), R^(3b), R^(3c) or R^(3d), provided that when R³ is boundto a heteroatom, then R³ is R^(3c) or R^(3d);

R^(3a) is —H, —F, —Cl, —Br, —I, —CF₃, —CN, N₃, —NO₂ or OR⁴;

R^(3b) is ═O, —O(R⁴), ═S, —N(R⁴), —N(O)(R⁴), —N(OR⁴), —N(O)(OR⁴), or—N(N(R⁴)(R⁴));

R^(3c) is —R⁴, —N(R⁴)(R⁴), —SR⁴, —S(O)R⁴, —S(O)₂R⁴, —S(O)(OR⁴),—S(O)₂(OR⁴), —OC(R^(3b))R⁴, —OC(R^(3b))OR⁴, —OC(R^(3b))(N(R⁴)(R⁴)),—SC(R^(3b))R⁴, —SC(R^(3b))OR⁴, —SC(R^(3b))(N(R⁴)(R⁴)),—N(R⁴)C(R^(3b))R⁴, —N(R⁴)C(R^(3b))OR⁴, —N(R⁴)C(R^(3b))(N(R⁴)(R⁴)), W³ or—R⁵W³;

R^(3d) is —C(R^(3b))R⁴, —C(R^(3b))OR⁴, —C(R^(3b))W³, —C(R^(3b))OW³ or—C(R^(3b))(N(R⁴)(R⁴));

R⁴ is —H, or an alkyl of 1 to 18 carbon atoms, alkenyl of 2 to 18 carbonatoms, or alkynyl of 2 to 18 carbon atoms;

R⁵ is alkylene of 1 to 18 carbon atoms, alkenylene of 2 to 18 carbonatoms, or alkynylene of 2 to 18 carbon atoms;

W³ is W⁴ or W⁵;

W⁴ is R⁶, —C(R^(3b))R⁶, —C(R^(3b))W⁵, —SO_(M2)R⁶, or —SO_(M2)W⁵ whereinR⁶ is R⁴ wherein each R⁴ is substituted with 0 to 3 R³ groups;

W⁵ is carbocycle or heterocycle wherein W⁵ is independently substitutedwith 0 to 3 R² groups; and

M2 is 0, 1 or 2;

and pharmaceutically acceptable salts thereof.

An embodiment of the present invention provides a compound of theFormula IA,

where Y^(1A) and Y^(1B) are as defined above.

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound of theformula,

An embodiment of the present invention provides a compound useful as anantiproliferative agent.

An embodiment of the present invention provides a compound useful as anapoptotic agent.

An embodiment of the present invention provides a compound useful as ananti-HPV agent.

An aspect of the present embodiment provides a compound useful as atopical anti-HPV agent.

An embodiment of the present invention provides a pharmaceuticalcomposition comprising an effective amount of a compound of Formula 1 ora pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

An aspect of the present embodiment provides a pharmaceuticalcomposition, where the composition is a gel composition.

Another aspect of the present embodiment provides a pharmaceuticalcomposition, where the composition is an ointment composition.

An embodiment of the present invention provides a pharmaceuticalcomposition comprising an effective amount of a compound of Formula 1 ora pharmaceutically acceptable salt thereof, and an effective amount ofat least one antiviral agent, and a pharmaceutically acceptable carrier.

An aspect of the present embodiment provides a pharmaceuticalcomposition, where the composition is a gel composition.

Another aspect of the present embodiment provides a pharmaceuticalcomposition, where the composition is an ointment composition.

DEFINITIONS

The term “PMEG” refers to the compound9-(2-phosphonylmethoxyethyl)guanine,

The term “PMEDAP” refers to the compound9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine,

The term “cprPMEDAP” refers to the compound9-(2-phosphonylmethoxyethyl)-2-amino-6-(cyclopropyl)purine,

“Bioavailability” is the degree to which the pharmaceutically activeagent becomes available to the target tissue after the agent'sintroduction into the body. Enhancement of the bioavailability of apharmaceutically active agent can provide a more efficient and effectivetreatment for patients because, for a given dose, more of thepharmaceutically active agent will be available at the targeted tissuesites.

The terms “phosphonate” and “phosphonate group” include functionalgroups or moieties within a molecule that comprises a phosphorous thatis 1) single-bonded to a carbon, 2) double-bonded to a heteroatom, 3)single-bonded to a heteroatom, and 4) single-bonded to anotherheteroatom, wherein each heteroatom can be the same or different. Theterms “phosphonate” and “phosphonate group” also include functionalgroups or moieties that comprise a phosphorous in the same oxidationstate as the phosphorous described above, as well as functional groupsor moieties that comprise a prodrug moiety that can separate from acompound so that the compound retains a phosphorous having thecharacteristics described above. For example, the terms “phosphonate”and “phosphonate group” include phosphonic acid, phosphonic monoester,phosphonic diester, phosphonamidate, and phosphonthioate functionalgroups. In one specific embodiment of the invention, the terms“phosphonate” and “phosphonate group” include functional groups ormoieties within a molecule that comprises a phosphorous that is 1)single-bonded to a carbon, 2) double-bonded to an oxygen, 3)single-bonded to an oxygen, and 4) single-bonded to another oxygen, aswell as functional groups or moieties that comprise a prodrug moietythat can separate from a compound so that the compound retains aphosphorous having such characteristics. In another specific embodimentof the invention, the terms “phosphonate” and “phosphonate group”include functional groups or moieties within a molecule that comprises aphosphorous that is 1) single-bonded to a carbon, 2) double-bonded to anoxygen, 3) single-bonded to an oxygen or nitrogen, and 4) single-bondedto another oxygen or nitrogen, as well as functional groups or moietiesthat comprise a prodrug moiety that can separate from a compound so thatthe compound retains a phosphorous having such characteristics.

Recipes and methods for determining stability of compounds in surrogategastrointestinal secretions are known. Compounds are defined herein asstable in the gastrointestinal tract where less than about 50 molepercent of the protected groups are deprotected in surrogate intestinalor gastric juice upon incubation for 1 hour at 37° C. Such compounds aresuitable for use in this embodiment. Note that simply because thecompounds are stable to the gastrointestinal tract does not mean thatthey cannot be hydrolyzed in viva. Prodrugs typically will be stable inthe digestive system but are substantially hydrolyzed to the parentaldrug in the digestive lumen, liver or other metabolic organ, or withincells in general.

The compounds of the invention can also exist as tautomeric isomers incertain cases. For example, ene-aminie tautomers can exist forimidazole, guanidine, amidine, and tetrazole systems and all theirpossible tautomeric forms are within the scope of the invention.

The term “prodrug” as used herein refers to any compound that whenadministered to a biological system generates the drug substance, i.e.active ingredient, as a result of spontaneous chemical reaction(s);enzyme catalyzed chemical reaction(s), photolysis, and/or metabolicchemical reaction(s). A prodrug is thus a covalently modified analog orlatent form of a therapeutically-active compound.

“Prodrug moiety” refers to a labile functional group which separatesfrom the active inhibitory compound during metabolism, systemically,inside a cell, by hydrolysis, enzymatic cleavage, or by some otherprocess (Bundgaard, Hans, “Design and Application of Prodrugs” in ATextbook of Drug Design and Development (1991), P. Krogsgaard-Larsen andH. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). Enzymeswhich are capable of an enzymatic activation mechanism with thephosphonate prodrug compounds of the invention include, but are notlimited to, amidases, esterases, microbial enzymes, phospholipases,cholinesterases, and phosphases. Prodrug moieties can serve to enhancesolubility, absorption and lipophilicity to optimize drug delivery,bioavailability and efficacy. A prodrug moiety may include an activemetabolite or drug itself.

Exemplary prodrug moieties include the hydrolytically sensitive orlabile acyloxymethyl esters —CH₂OC(═O)R and acyloxymethyl carbonates—CH₂OC(═O)OR where R in this instance is C₁-C₆ alkyl, C₁-C₆ substitutedalkyl, C₆-C₂₀ aryl or C₆-C₂₀ substituted aryl. The acyloxyalkyl esterwas first used as a prodrug strategy for carboxylic acids and thenapplied to phosphates and phosphonates by Farquhar et al. (1983) J.Pharm. Sci. 72: 324; also U.S. Pat. Nos. 4,816,570, 4,968,788, 5,663,159and 5,792,756. Subsequently, the acyloxyalkyl ester was used to deliverphosphonic acids across cell membranes and to enhance oralbioavailability. A close variant of the acyloxyalkyl ester, thealkoxycarbonyloxyalkyl ester (carbonate), may also enhance oralbioavailability as a prodrug moiety in the compounds of the combinationsof the invention. An exemplary acyloxymethyl ester isisopropylcarbonyloxymethoxy, —OCH₂OC(═O)C(CH₃)₂. An exemplaryacyloxymethyl carbonate prodrug moiety is isopropylcarbonyloxymethylcarbonate, HOC(═O)OCH₂OC(═O)C(CH₃)₂.

The phosphonate group may be a phosphonate prodrug moiety. The prodrugmoiety may be sensitive to hydrolysis, such as, but not limited to aisopropylcarbonyl-oxymethoxy or isopropylcarbonyloxymethyl carbonategroup. Alternatively, the prodrug moiety may be sensitive to enzymaticpotentiated cleavage, such as a lactate ester or a phosphonamidate-estergroup.

Aryl esters of phosphorus groups, especially phenyl esters, are reportedto enhance oral bioavailability (De Lombaert et al. (1994) J. Med. Chem.37:498). Phenyl esters containing a carboxylic ester ortho to thephosphate have also been described (Khamnei and Torrence, (1996) J. Med.Chem. 39:4109-4115). Benzyl esters are reported to generate the parentphosphonic acid. In some cases, substituents at the ortho- orpara-position may accelerate the hydrolysis. Benzyl analogs with anacylated phenol or an alkylated phenol may generate the phenoliccompound through the action of enzymes, e.g., esterases, oxidases, etc.,which in turn undergoes cleavage at the benzylic C—O bond to generatethe phosphoric acid and the quinone methide intermediate. Examples ofthis class of prodrugs are described by Mitchell et al. (1992) J. Chem.Soc. Perkin Trans. II 2345; Glazier WO 91/19721. Still other benzylicprodrugs have been described containing a carboxylic ester-containinggroup attached to the benzylic methylene (Glazier WO 91/19721).Thio-containing prodrugs are reported to be useful for the intracellulardelivery of phosphonate drugs. These proesters contain an ethylthiogroup in which the thiol group is either esterified with an acyl groupor combined with another thiol group to form a disulfide.Deesterification or reduction of the disulfide generates the free thiointermediate which subsequently breaks down to the phosphoric acid andepisulfide (Puech et al. (1993) Antiviral Res., 22: 155-174; Benzaria etal. (1996) J. Med. Chem. 39: 4958). Cyclic phosphonate esters have alsobeen described as prodrugs of phosphorus-containing compounds (Erion etal., U.S. Pat. No. 6,312,662).

“Protecting group” refers to a moiety of a compound that masks or altersthe properties of a functional group or the properties of the compoundas a whole. Chemical protecting groups and strategies forprotection/deprotection are well known in the art. See e.g., ProtectiveGroups in Organic Chemistry, Theodora W. Greene, John Wiley & Sons,Inc., New York, 1991. Protecting groups are often utilized to mask thereactivity of certain functional groups, to assist in the efficiency ofdesired chemical reactions, e.g., making and breaking chemical bonds inan ordered and planned fashion. Protection of functional groups of acompound alters other physical properties besides the reactivity of theprotected functional group, such as the polarity, lipophilicity(hydrophobicity), and other properties which can be measured by commonanalytical tools. Chemically protected intermediates may themselves bebiologically active or inactive.

Protected compounds may also exhibit altered, and in some cases,optimized properties in vitro and in vivo, such as passage throughcellular membranes and resistance to enzymatic degradation orsequestration. In this role, protected compounds with intendedtherapeutic effects may be referred to as prodrugs.

Another function of a protecting group is to convert the parental druginto a prodrug, whereby the parental drug is released upon conversion ofthe prodrug in vivo. Because active prodrugs may be absorbed moreeffectively than the parental drug, prodrugs may possess greater potencyin vivo than the parental drug. Protecting groups are removed either invitro, in the instance of chemical intermediates, or in vivo, in thecase of prodrugs. With chemical intermediates, it is not particularlyimportant that the resulting products after deprotection, e.g.,alcohols, be physiologically acceptable, although in general it is moredesirable if the products are pharmacologically innocuous.

Any reference to any of the compounds of the invention also includes areference to a physiologically acceptable salt thereof. Examples ofphysiologically acceptable salts of the compounds of the inventioninclude salts derived from an appropriate base, such as an alkali metal(for example, sodium), an alkaline earth (for example, magnesium),ammonium and NX₄ ₊ (wherein X is C₁-C₄ alkyl). Physiologicallyacceptable salts of an hydrogen atom or an amino group include salts oforganic carboxylic acids such as acetic, benzoic, lactic, fumaric,tartaric, maleic, malonic, malic, isethionic, lactobionic and succinicacids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic,benzenesulfonic and p-toluenesulfonic acids; and inorganic acids, suchas hydrochloric, sulfuric, phosphoric and sulfamic acids.Physiologically acceptable salts of a compound of a hydroxy groupinclude the anion of said compound in combination with a suitable cationsuch as Na⁺ and NX₄ ₊ (wherein X is independently selected from WI or aC₁-C₄ alkyl group).

As used herein, the term “gel” refers to semisolid systems consisting ofeither suspensions made up of small inorganic particles or large organicmolecules enclosing and interpenetrated by a liquid. Where the gel massconsists of floccules of small particles, the gel is classified as atwo-phase system and is sometimes called a magma. Aluminum Hydroxide Geland Bentonite Magma are examples of two-phase systems. Single-phase gelsconsist of organic macromolecules uniformly distributed throughout aliquid in such a manner that no apparent boundaries exist between thedispersed macromolecules and the liquid. Examples of such gels areCarboxymethylcellulose Sodium and Tragacanth. Although gels are commonlyaqueous, alcohols and oils may be used as a continuous phase.

As used herein the term “ointment” refers to a semisolid preparation forexternal application of such consistency that they may be readilyapplied to skin by injunction. They should be of such composition thatthey soften but not necessarily melt when applied to the body. Theyserve as vehicles for the topical application of medicinal substancesand also function as protectives and emollients for the skin.

For therapeutic use, salts of active ingredients of the compounds of theinvention will be physiologically acceptable, i.e. they will be saltsderived from a physiologically acceptable acid or base. However, saltsof acids or bases which are not physiologically acceptable may also finduse, for example, in the preparation or purification of aphysiologically acceptable compound. All salts, whether or not derivedform a physiologically acceptable acid or base, are within the scope ofthe present invention.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto, ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to,acetylenic (—C≡CH) and propargyl (—CH₂C≡CH),

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to, methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to, 1,2-ethylene(—CH═H—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to, acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. The arylalkyl group comprises 6to 20 carbon atoms, e.g., the alkyl moiety, including alkyl, alkenyl oralkynyl groups, of the arylalkyl group is 1 to 6 carbon atoms and thearyl moiety is 5 to 14 carbon atoms.

“Substituted alkyl”, “substituted aryl”, and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a non-hydrogensubstituent. Typical substituents include, but are not limited to, -X,-R, —O—, —OR, —SR, —S—, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O,—NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NRR —S(═O)₂O⁻,—S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NHR, —S(═O)R,—OP(═O)O₂RR—P(═O)I₂RR—P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R, —C(═O)X, —C(S)R,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR,—C(NR)NRR, where each X is independently a halogen: F, Cl, Br, or I; andeach R is independently —H, alkyl, aryl, heterocycle, protecting groupor prodrug moiety. Alkylene, alkenylene, and alkynylene groups may alsobe similarly substituted.

“Heterocycle” as used herein includes by way of example and notlimitation these heterocycles described in Paquette, Leo A.; Principlesof Modern Heterocyclic Chemistry (W. A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry ofHeterocyclic Compounds, A Series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment of theinvention “heterocycle” includes a “carbocycle” as defined herein,wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replacedwith a heteroatom (e.g. O, N, or S).

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolentyl, quinolinylisoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,isatinoyl, and bis-tetrahydrofuranyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

“Carbocycle” refers to a saturated, unsaturated or aromatic ring having3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle,and up to about 20 carbon atoms as a polycycle. Monocyclic carbocycleshave 3 to 6 ring atoms, still more typically 5 or 6 ring atoms. Bicycliccarbocycles have 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as abicyclo [5,6] or [6,6] system. Examples of monocyclic carbocyclesinclude cyclopropyl (cPropyl), cyclobutyl (cButyl), cyclopentyl(cPentyl), 1-cyclopent-1-enyl, 1-cyclopent-2-enyl 1-cyclopent-3-enyl,cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,phenyl, spiryl and naphthyl.

“Linker” or “link” refers to a chemical moiety comprising a covalentbond or a chain or group of atoms that covalently attaches a phosphonategroup to a drug. Linkers include moieties such as: repeating units ofalkyloxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino(e.g., polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

As used herein the term “Aba” refers to a divalent moiety of2-aminobutanoic acid,

where the points of attachment are designated by the “*”.

As used herein the term “Ala” refers to a divalent moiety of alanine,

where the points of attachment are designated by the “*”.

As used herein the term “Phe” refers to a divalent moiety of alanine,

where the points of attachment are designated by the “*”.

As used herein the term “Ala” refers to a divalent moiety of alanine,

where the points of attachment are designated by the “*”.

As used herein the term “POC” refers to the divalent moiety ofhydroxymethyl isopropyl carbonate,

where the point of attachment is designated by the “*”.

Substitutent groups Y^(1A) and Y^(1B) can be described usingnomenclature that incorporates the aforementioned divalent amino acidmoieties and alkyl moieties, such as in Table 80-3.

For example, the compound of the formula,

can be described using the nomenclature of Formula I, where Y^(1A) andY^(1B) are —N(R^(X)), where R^(X) is R², where R² is R⁴ substituted withR^(3d), where R⁴ is ethyl substituted with R^(3d) further where R^(3d)is —C(R^(3b))OW³, where R^(3b) is ═O, where W³ is W⁵, where W⁵ is acarbocycle, where R⁴ is propyl substituted with R^(3d), where R^(3d) is—C(R^(3b))OR⁴, where R^(3b) is ═O, and where R⁴ is ethyl. Alternatively,said compound can be described, as in Table 80-3, as Formula I, whereY^(1A) and Y^(1B) are “Aba-Et”, which describes the moiety (where the“*” indicates the point of attachment),

which is “Aba” linked to “Et” (ethyl).

For example, the compound of the formula,

can be described using the nomenclature of Formula I, where Y^(1A) andY^(1B) are —N(R^(X)), where R^(X) is R², where R² is R⁴ substituted withR^(3d), where R⁴ is ethyl substituted with R^(3d), where R^(3d) is—C(R^(3b))OR⁴, where R^(3b) is ═O, and where R⁴ is n-propyl.Alternatively, said compound can be described, as in Table 80-3, asFormula I, where Y^(1A) and Y^(1B) are “Ala-nPr”, which describes themoiety (where the “*” indicates the point of attachment),

which is “Ala” linked to “nPr” (n-propyl).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The term “treatment” or “treating,” to the extent it relates to adisease or condition includes preventing the disease or condition fromoccurring, inhibiting the disease or condition, eliminating the diseaseor condition, and/or relieving one or more symptoms of the disease orcondition.

The term “antiproliferative” refers to activities used to, or tending toinhibit cell growth, such as antiproliferative effects on tumor cells,or antiproliferative effects on virally infected cells.

The terms “apoptosis” refers to one of the main types of programmed celldeath. As such, it is a process of deliberate suicide by an unwantedcell in a multicellular organism. In contrast to necrosis, which is aform of cell death that results from acute tissue injury, apoptosis iscarried out in an ordered process that generally confers advantagesduring an organism's life cycle. Apoptosis is a type of cell death inwhich the cell uses specialized cellular machinery to kill itself; acell suicide mechanism that enables metazoans to control cell number andeliminate cells that threaten the animal's survival. Apoptosis canoccur, for instance, when a cell is damaged beyond repair, or infectedwith a virus. The stimuli for apoptosis can come from the cell itself,from its surrounding tissue or from a cell that is part of the immunesystem, it can be chemical, biological or physical. The related term“apoptitic” refers to the process of apoptosis.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and 1 or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or 1 meaning that the compound is levorotatory. A compoundprefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity

Protecting Groups

In the context of the present invention, protecting groups includeprodrug moieties and chemical protecting groups.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds of the invention. For the most part the decision as to whichgroups to protect, when to do so, and the nature of the chemicalprotecting group “PG” will be dependent upon the chemistry of thereaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis. The PG groups do not need to be, and generally are not, thesame if the compound is substituted with multiple PG. In general, PGwill be used to protect functional groups such as carboxyl, hydroxyl,thio, or amino groups and to thus prevent side reactions or to otherwisefacilitate the synthetic efficiency. The order of deprotection to yieldfree, deprotected groups is dependent upon the intended direction of thesynthesis and the reaction conditions to be encountered, and may occurin any order as determined by the artisan.

Various functional groups of the compounds of the invention may beprotected. For example, protecting groups for —OH groups (whetherhydroxyl, carboxylic acid, phosphonic acid, or other functions) include“ether- or ester-forming groups”. Ether- or ester-forming groups arecapable of functioning as chemical protecting groups in the syntheticschemes set forth herein. However, some hydroxyl and thio protectinggroups are neither ether- nor ester-forming groups, as will beunderstood by those skilled in the art, and are included with amides,discussed below.

A very large number of hydroxyl protecting groups and amide-forminggroups and corresponding chemical cleavage reactions are described inProtective Groups in Organic Synthesis, Theodora W. Greene (John Wiley &Sons, Inc., New York, 1991, ISBN 0-471-62301-6) (“Greene”). See alsoKocienski, Philip J.; Protecting Groups (Georg Thieme Verlag Stuttgart,New York, 1994), which is incorporated by reference in its entiretyherein. In particular Chapter 1, Protecting Groups: An Overview, pages1-20, Chapter 2, Hydroxyl Protecting Groups, pages 21-94, Chapter 3,Diol Protecting Groups, pages 95-117, Chapter 4, Carboxyl ProtectingGroups, pages 118-154, Chapter 5, Carbonyl Protecting Groups, pages155-184. For protecting groups for carboxylic acid, phosphonic acid,phosphonate, sulfonic acid and other protecting groups for acids seeGreene as set forth below. Such groups include by way of example and notlimitation, esters, amides, hydrazides, and the like.

Ether- and Ester-Forming Protecting Groups

Ester-forming groups include: (1) phosphonate ester-forming groups, suchas phosphonamidate esters, phosphorothioate esters, phosphonate esters,and phosphon-bis-amidates; (2) carboxyl ester-forming groups, and (3)sulphur ester-forming groups, such as sulphonate, sulfate, andsulfinate.

The phosphonate moieties of the compounds of the invention may or maynot be prodrug moieties, i.e. they may or may be susceptible tohydrolytic or enzymatic cleavage or modification. Certain phosphonatemoieties are stable under most or nearly all metabolic conditions. Forexample, a dialkylphosphonate, where the alkyl groups are two or morecarbons, may have appreciable stability in vivo due to a slow rate ofhydrolysis.

Salts and Hydrates

The compositions of this invention optionally comprise salts of thecompounds herein, especially pharmaceutically acceptable nontoxic saltscontaining, for example, Na⁺, Li⁺, K⁺, Ca⁺⁺ and Mg⁺⁺. Such salts mayinclude those derived by combination of appropriate cations such asalkali and alkaline earth metal ions or ammonium and quaternary aminoions with an acid anion moiety. Monovalent salts are preferred if awater soluble salt is desired.

Metal salts typically are prepared by reacting a compound of thisinvention with a metal hydroxide. Examples of metal salts which areprepared in this way are salts containing Li⁺, Na⁺, and K⁺. A lesssoluble metal salt can be precipitated from the solution of a moresoluble salt by addition of the suitable metal compound.

In addition, salts may be formed from acid addition of certain organicand inorganic acids, e.g., HCl, HBr, H₂SO₄, or organic sulfonic acids,to basic centers, or to acidic groups. Finally, it is to be understoodthat the compositions herein comprise compounds of the invention intheir un-ionized, as well as zwitterionic form, and combinations withstoichiometric amounts of water as in hydrates.

Also included within the scope of this invention are the salts of theparental compounds with one or more amino acids. Any of the amino acidsdescribed above are suitable, especially the naturally-occurring aminoacids found as protein components, although the amino acid typically isone bearing a side chain with a basic or acidic group, e.g., lysine,arginine or glutamic acid, or a neutral group such as glycine, serine,threonine, alanine, isoleucine, or leucine.

Methods of Inhibition of HPV

Another aspect of the invention relates to methods of inhibiting theactivity of HPV comprising the step of treating a sample suspected ofcontaining HPV with a compound of the invention.

Compositions of the invention act as inhibitors of HPV, as intermediatesfor such inhibitors or have other utilities as described below.

The treating step of the invention comprises adding the composition ofthe invention to the sample or it comprises adding a precursor of thecomposition to the sample. The addition step comprises any method ofadministration as described above.

If desired, the activity of HPV after application of the composition canbe observed by any method including direct and indirect methods ofdetecting HPV activity. Quantitative, qualitative, and semi quantitativemethods of determining HPV activity are all contemplated. Typically oneof the screening methods described above are applied, however, any othermethod such as observation of the physiological properties of a livingorganism are also applicable.

Screens for HPV Inhibitors

Compounds and compositions of the invention are screened for therapeuticutility by measuring the EC₅₀, that is the concentration of compoundthat achieves 50% inhibition of cell growth. The ratio of EC₅₀ inHPV-uninfected and infected cells provides a measure of the selectivityof the compound for the virus infected cells. The protocols used toobtain these measures are taught in the Examples.

Pharmaceutical Formulations and Routes of Administration.

The compounds of this invention are formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Tablets will contain excipients, glidants, fillers, bindersand the like. Aqueous formulations are prepared in sterile form, andwhen intended for delivery by other than oral administration generallywill be isotopic. All formulations will optionally contain excipientssuch as those set forth in the “Handbook of Pharmaceutical Excipients”(1986). Excipients include ascorbic acid and other antioxidants,chelating agents such as EDTA, carbohydrates such as dextrin,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations ranges from about 3 to about 11, but isordinarily about 7 to 10.

One or more compounds of the invention (herein referred to in thiscontext as the active ingredients) are administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, rectal, nasal topical (including buccal and sublingual), vaginaland parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural), and the like. It will beappreciated that the preferred route may vary with the condition of therecipient. An advantage of the compounds of this invention is that theyare orally bioavailable and can be dosed orally.

While it is possible for the active ingredients to be administered aloneit may be preferable to present them as pharmaceutical formulations. Theformulations, both for veterinary and for human use, of the inventioncomprise at least one active ingredient, as above defined, together withone or more acceptable carriers therefore and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand physiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the invention suitable for oral administration areprepared as discrete units such as capsules, cachets or tablets eachcontaining a predetermined amount of the active ingredient; as a powderor granules; as solution or a suspension in an aqueous liquid or anon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredientthere from.

For infections of the eye or other external tissues e.g. mouth and skin,the formulations are preferably applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredients) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the active ingredients may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredients may be formulated in a cream with an oil-in-watercream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of a polyhydric alcohol i.e. an alcohol havingtwo or more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulphoxide and relatedanalogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. The cream should preferablybe a non-greasy, non-staining and washable product with suitableconsistency to avoid leakage from tubes or other containers. Straight orbranched chain, mono- or dibasic alkyl esters such as di-isoadipate,isocetyl stearate, propylene glycol diester of coconut fatty acids,isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils are used.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of influenza A or B infections as describedbelow.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers are asknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations are presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Compounds of the invention are used to provide controlled releasepharmaceutical formulations containing as active ingredient one or morecompounds of the invention (“controlled release formulations”) in whichthe release of the active ingredient are controlled and regulated toallow less frequency dosing or to improve the pharmacokinetic ortoxicity profile of a given active ingredient.

Effective dose of active ingredient depends at least on the nature ofthe condition being treated, toxicity, whether the compound is beingused prophylactically (lower doses) or against an active influenzainfection, the method of delivery, and the pharmaceutical formulation,and will be determined by the clinician using conventional doseescalation studies. It can be expected to be from about 0.0001 to about100 mg/kg body weight per day; typically, from about 0.01 to about 10mg/kg body weight per day; more typically, from about 0.01 to about 5mg/kg body weight per day; most typically, from about 0.05 to about 0.5mg/kg body weight per day. For example, for inhalation the dailycandidate dose for an adult human of approximately 70 kg body weightwill range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, andmay take the form of single or multiple doses.

Active ingredients of the invention are also used in combination withother active ingredients. Such combinations are selected based on thecondition to be treated, cross-reactivities of ingredients andpharmaco-properties of the combination. For example, when treating viralinfections of the respiratory system, in particular influenza infection,the compositions of the invention are combined with antivirals (such asamantidine, rimantadine and ribavirin), mucolytics, expectorants,bronchialdilators, antibiotics, antipyretics, or analgesics. Ordinarily,antibiotics, antipyretics, and analgesics are administered together withthe compounds of this invention.

Metabolites of the Compounds of the Invention

The present invention also provides the in vivo metabolic products ofthe compounds described herein, to the extent such products are noveland unobvious over the prior art. Such products may result for examplefrom the oxidation, reduction, hydrolysis, amidation, esterification andthe like of the administered compound, primarily due to enzymaticprocesses. Accordingly, the invention includes novel and unobviouscompounds produced by a process comprising contacting a compound of thisinvention with a mammal for a period of time sufficient to yield ametabolic product thereof. Such products typically are identified bypreparing a radiolabeled (e.g. C¹⁴ or H³) compound of the invention,administering it parenterally in a detectable dose (e.g. greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g. by MSor NMR analysis. In general, analysis of metabolites is done in the sameway as conventional drug metabolism studies well-known to those skilledin the art. The conversion products, so long as they are not otherwisefound in vivo, are useful in diagnostic assays for therapeutic dosing ofthe compounds of the invention even if they possess no neuraminidaseinhibitory activity of their own.

Additional Uses for the Compounds of this Invention.

The compounds of this invention, or the biologically active substancesproduced from these compounds by hydrolysis or metabolism in vivo, areused as immunogens or for conjugation to proteins, whereby they serve ascomponents of immunogenic compositions to prepare antibodies capable ofbinding specifically to the protein, to the compounds or to theirmetabolic products which retain immunologically recognized epitopes(sites of antibody binding). The immunogenic compositions therefore areuseful as intermediates in the preparation of antibodies for use indiagnostic, quality control, or the like, methods or in assays for thecompounds or their novel metabolic products. The compounds are usefulfor raising antibodies against otherwise non-immunogenic polypeptides,in that the compounds serve as haptenic sites stimulating an immuneresponse that cross-reacts with the unmodified conjugated protein.

The hydrolysis products of interest include products of the hydrolysisof the protected acidic and basic groups discussed above. As notedabove, the acidic or basic amides comprising immunogenic polypeptidessuch as albumin or keyhole limpet hemocyanin generally are useful asimmunogens. The metabolic products described above may retain asubstantial degree of immunological cross reactivity with the compoundsof the invention. Thus, the antibodies of this invention will be capableof binding to the unprotected compounds of the invention without bindingto the protected compounds; alternatively the metabolic products, willbe capable of binding to the protected compounds and/or the metabolicproducts without binding to the protected compounds of the invention, orwill be capable of binding specifically to any one or all three. Theantibodies desirably will not substantially cross-react withnaturally-occurring materials. Substantial cross-reactivity isreactivity under specific assay conditions for specific analytessufficient to interfere with the assay results.

The immunogens of this invention contain the compound of this inventionpresenting the desired epitope in association with all immunogenicsubstance. Within the context of the invention such association meanscovalent bonding to form an immunogenic conjugate (when applicable) or amixture of non-covalently bonded materials, or a combination of theabove. Immunogenic substances include adjuvants such as Freund'sadjuvant, immunogenic proteins such as viral, bacterial, yeast, plantand animal polypeptides, in particular keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin or soybean trypsin inhibitor, andimmunogenic polysaccharides. Typically, the compound having thestructure of the desired epitope is covalently conjugated to animmunogenic polypeptide or polysaccharide by the use of a polyfunctional(ordinarily bifunctional) cross-linking agent. Methods for themanufacture of hapten immunogens are conventional per se, and any of themethods used heretofore for conjugating haptens to immunogenicpolypeptides or the like are suitably employed here as well, taking intoaccount the functional groups on the precursors or hydrolytic productswhich are available for cross-linking and the likelihood of producingantibodies specific to the epitope in question as opposed to theimmunogenic substance.

Typically the polypeptide is conjugated to a site on the compound of theinvention distant from the epitope to be recognized.

The conjugates are prepared in conventional fashion. For example, thecross-linking agents N-hydroxysuccinimide, succinic anhydride oralkN═C═Nalk are useful in preparing the conjugates of this invention.The conjugates comprise a compound of the invention attached by a bondor a linking group of 1-100, typically, 1-25, more typically 1-10 carbonatoms to the immunogenic substance. The conjugates are separated fromstarting materials and by products using chromatography or the like, andthen are sterile filtered and vialed for storage.

Animals are typically immunized against the immunogenic conjugates orderivatives and antisera or monoclonal antibodies prepared inconventional fashion.

The compounds of this invention are useful as linkers or spacers inpreparing affinity absorption matrices, immobilized enzymes for processcontrol or immunoassay reagents. The compounds herein contain amultiplicity of functional groups that are suitable as sites forcross-linking desired substances. For example, it is conventional tolink affinity reagents such as hormones, peptides, antibodies, drugs,and the like to insoluble substrates. These insolubilized reagents areemployed in known fashion to absorb binding partners for the affinityreagents from manufactured preparations, diagnostic samples and otherimpure mixtures. Similarly, immobilized enzymes are used to performcatalytic conversions with facile recovery of enzyme. Bifunctionalcompounds are commonly used to link analytes to detectable groups inpreparing diagnostic reagents.

Screening assays preferably use cells from particular tissues that aresusceptible to HPV infection. Assays known in the art are suitable fordetermining in vivo bioavailability including intestinal lumenstability, cell permeation, liver homogenate stability and plasmastability assays. However, even if the ester, amide or other protectedderivatives are not converted in vivo to the free carboxyl, amino orhydroxyl groups, they remain useful as chemical intermediates.

Utility for the present invention was taught using antiproliferationassays. Antiproliferation assays measure effect of compounds onproliferation of cultured cells. Cells are cultured for 7 days in thepresence of various concentrations of compounds. On the 7^(th) day,cells are stained with dye, and intensity of staining (proportional tocell number) is measured by spectrophotometer. Data are plotted againstcompound concentrations, fitted to the sigmoid dose response curve, fromwhich the compound concentration that reduces cell proliferation rate by500% (50% effective concentration or EC₅₀) is determined. Activecompounds in antiproliferation assays may be cytostatic (inhibit celldivision) and/or cytocidal (kill cells). By performing antiproliferationassays in HPV positive cancer cells and normal cells, we identifycompounds that inhibit proliferation of HPV positive cancer cells moreefficiently than cells from normal human tissues.

Exemplary Methods of Making the Compounds of the Invention.

The invention also relates to methods of making the compositions of theinvention. The compositions are prepared by any of the applicabletechniques of organic synthesis. Many such techniques are well known inthe art. However, many of the known techniques are elaborated in“Compendium of Organic Synthetic Methods” (John Wiley & Sons, New York),Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, jr., 1980; Vol. 5, Leroy C. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as March, J., “AdvancedOrganic Chemistry, Third Edition”, (John Wiley & Sons, New York, 1985),“Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency inModern Organic Chemistry. In 9 Volumes”, Barry M. Trost, Editor-in-Chief(Pergamon Press, New York, 1993 printing).

A number of exemplary methods for the preparation of the compositions ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, workup procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic, and reactiontimes will be 10 seconds to 10 days. Workup typically consists ofquenching any unreacted reagents followed by partition between awater/organic layer system (extraction) and separating the layercontaining the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C., solvents are typically aprotic for reductions and may beeither protic or aprotic for oxidations. Reaction times are adjusted toachieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions).

Standard synthetic techniques such as azeotropic removal of reactionby-products and use of anhydrous reaction conditions (e.g. inert gasenvironments) are common in the art and will be applied when applicable.

Exemplary methods of preparing the compounds of the invention are shownin the schemes below. Detailed descriptions of the methods are found inthe Experimental section below, and are referenced to the specificschemes.

Schemes

Each of the products of the following processes is optionally separated,isolated, and/or purified prior to its use in subsequent processes.

The terms “treated”, “treating”, “treatment”, and the like, when used inthe context of a chemical process, protocol, or preparation meancontacting, mixing, reacting, allowing to react, bringing into contact,and other terms common in the art for indicating that one or morechemical entities is treated in such a manner as to convert it to one ormore other chemical entities. This means that “treating compound onewith compound two” is synonymous with “allowing compound one to reactwith compound two”, “contacting compound one with compound two”,“reacting compound one with compound two”, and other expressions commonin the art of organic synthesis for reasonably indicating that compoundone was “treated”, “reacted”, “allowed to react”, etc., with compoundtwo.

In the context of a chemical process, protocol, or preparation,“treating” indicates the reasonable and usual manner in which organicchemicals are allowed to react. Normal concentrations (0.01M to 10M,typically 0.1M to 1M), temperatures (−100° C. to 250° C., typically −78°C. to 150° C., more typically −78° C. to 100° C., still more typically0° C. to 100° C.), reaction vessels (typically glass, plastic, metal),solvents, pressures, atmospheres (typically air for oxygen and waterinsensitive reactions or nitrogen or argon for oxygen or water sensitivereactions), etc., are intended unless otherwise indicated. The knowledgeof similar reactions known in the art of organic synthesis is used inselecting the conditions and apparatus for “treating” in a givenprocess. In particular, one of ordinary skill in the art of organicsynthesis selects conditions and apparatus reasonably expected tosuccessfully carry out the chemical reactions of the described processesbased on the knowledge in the art.

Modifications of each of the above scheme(s) leads to various analogs ofthe specific exemplary materials produced above. The above citedcitations describing suitable methods of organic synthesis areapplicable to such modifications.

In each of the above exemplary schemes it may be advantageous toseparate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified (hereinafter separated) to the desired degreeof homogeneity by the techniques common in the art. Typically suchseparations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example, size exclusion or ion exchange chromatography,high, medium, or low pressure liquid chromatography, small scale andpreparative thin or thick layer chromatography, as well as techniques ofsmall scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved, for example, boiling point, and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

All literature and patent citations above are hereby expresslyincorporated by reference at the locations of their citation.Specifically cited sections or pages of the above cited works areincorporated by reference with specificity. The invention has beendescribed in detail sufficient to allow one of ordinary skill in the artto make and use the subject matter of the following claims. It isapparent that certain modifications of the methods and compositions ofthe following claims can be made within the scope and spirit of theinvention. The following Examples are provided to exemplify the presentinvention, and in no means can be construed to limit the presentinvention.

EXAMPLES General

Some Examples have been performed multiple times. In repeated Examples,reaction conditions such as time, temperature, concentration and thelike, and yields were within normal experimental ranges. In repeatedExamples where significant modifications were made, these have beennoted where the results varied significantly from those described. InExamples where different starting materials were used, these are noted.When the repeated Examples refer to a “corresponding” analog of acompound, such as a “corresponding ethyl ester”, this intends that anotherwise present group, in this case typically a methyl ester, is takento be the same group modified as indicated.

Examples 1 to 35 Refer to Schemes 1 to 9 Above Example 1

Acetoxyethyloxymethylchloride 1: A 5 L three-neck flask was fitted withmechanical stirrer, thermometer, 500 mL additional funnel and argonpurged. 1,3-Dioxalane (140 mL, 2.00 mol) in anhydrous Et₂O (800 mL) and1.0 M ZlnCl₂/Et₂O (7.5 mL, 0.007 mol) were added. A solution of acetylchloride (157 mL, 2.20 mol) in Et₂O (200 mL) was added dropwise throughan additional funnel over 20 min. A cold water bath was used to maintaintemperature between 19-27° C. throughout. Continue stirring withoutexternal cooling for 4 h, reaction self heating at 20-25° C. for about 1h. A clear, colorless solution retained under argon overnight. Stood for3 days and formed an orange solution. Strip Et₂O on rotavap (wateraspirator) until no more distilled at 35° C. bath. A quantitative yieldof product 318 g (theoretical yield 306 g) was obtained.

Example 2

Diisopropyl Phosphonate 2: A 500 mL three-neck flask was charged withthe crude chloromethylether 1 (317 g, 2.00 mol). Triisopropylphosphite(494 mL) was added dropwise through an additional funnel while heatingin a 125° C. oil bath and stirring vigorously. Collect 2-chloropropanedistillate via short-path head in a dry ice cooled receiver, argonblanket, collected 140 g distillate (theoretical 157 g). Phosphiteblanched reaction to yellow, continue heating another 2 h at 125° C. oilbath, then arrange for vacuum distillation using a vacuum pump.Distilled a yellow front cut (140 g, head to 135° C., bottom to 190°C.), then changed to clean receiver. Main fraction was collected at headtemperature of 178-187° C. (mostly 185-187° C.) with vacuum unknown atbath temperature of 222-228° C. 258 g of the product 2 was given (47%yield from 1,3-dioxolane).

Example 3

Alcohol 3: A solution of 2 (125 g, 0.443 mol) in absolute MeOH (440 mL)was treated with concentrated MCl (11.2 mL, 0.112 mol) and heated toreflux for 6 h under Argon. Strip MeOH on rotavap (water aspirator) to55° C. leaving 115 g of a clear oil which was co-evaporated with toluene(2×200 mL). The crude product was dried under vacuum to give an oil (102g, 96%).

Example 4

Diisopropyl Phosphonate 4: A solution of triphenylphosphine (25.57 g,97.5 mmol) and alcohol 3 (18 g, 75 mmol.) in DMF (120 mL) was treatedwith 6-chloropurine (12.72 g, 75 mmol.) and cooled to −15° C. A solutionof diisopropyl azodicarboxylate (16.68 g, 82.5 mmol) in DMF (50 mL) wasadded dropwise through an additional funnel over 80 min. The reactionmixture was kept at −15° C. for 2 h and then warmed to room temperatureand stirred for an additional 2 h. A cloudy reaction mixture turned tobe a bright yellow solution. The reaction solvent was evaporated underreduced pressure, co-evaporated with toluene (3×), and dried undervacuum overnight prior to purification. The crude product was purifiedby column chromatography on silica gel (5% MeOH/CH₂Cl₂) to give thediisopropyl phosphonate (18.52 g, 63%) as a white solid: ¹H NMR (CDCl₃)δ 7.95 (s, 1H), 4.70 (m, 2H), 4.31 (m, 2H), 3.93 (m, 2H), 3.73 (m, 2H),1.29 (m, 12H); ³¹P NMR (CDCl₃) δ 18.42.

Example 5

Diisopropyl Phosphonate 5: A mixture of 4 (11.00 g, 28.08 mmol) andcyclopropylamine (4.86 g, 85.16 mmol) in CH₃CN (80 mL) was placed in areaction bomb and heated to 100° C. for 4 h. The reaction mixture wascooled to room temperature and concentrated under reduced pressure. Theproduct was partitioned between 15% MeOH/CH₂Cl₂ (3×) and brine, driedwith Na₂SO₄, filtered, and concentrated. The crude product was purifiedby column chromatography on silica gel (5% MeOH/CH₂Cl₂) to give 5 (10.42g, 90%) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.59 (s, 1H), 5.83(broad, s, 1H), 4.88 (broad, s, 2H), 4.70 (m, 2H), 4.21 (m, 2H), 3.88(m, 2H), 3.72 (d, J=8.4 Hz, 2H), 3.03 broad, s, 1H), 1.28 (m, 12H), 0.84(m, 2H), 0.60 (m, 2H); ³¹P NMR (CDCl₃) δ 18.63.

Example 6

cPrPMEDAP 6: A solution of 5 (11.00 g, 26.67 mmol) in anhydrous CH₃CN(120 mL) was treated with bromotrimethylsilane (21.1 mL, 160.02 mmol).The reaction was protected from light by wrapping the flask withaluminum foil. The reaction mixture was stirred at room temperatureovernight. The volatiles were evaporated under reduced pressure. Theresidue was dissolved in H₂O (250 mL) and pH was adjusted to 9 withammonium hydroxide. The reaction mixture was concentrated and a yellowsolid was obtained. The solid was dissolved in H₂O (30 mL) and pH wasadjusted to 2 with 10% HCl. Fine solid was collected and dried undervacuum to give 6 (7.88 g, 90%) as a white solid.

Example 7

Monophosphonic Acid Hydrochloride 7: A mixture of acid 6 (3.00 g, 9.15mmol) and DMF (0.1 mL) in sulfolane (9.2 mL) was heated to 70° C.Thionylchloride (1.66 mL, 22.76 mmol) was added dropwise over a periodof 1 h. The temperature was increased to 90° C. and TMSOPh (1.74 mL,9.61 mmol) was added and stirred for 1 h. The reaction mixture wascooled to room temperature overnight. The reaction mixture was addeddropwise to well-stirred, ice-cold acetone (100 mL). The product wasprecipitated out. The solid was filtered under Ar, washed with coldacetone (100 mL), dried under vacuum to give the monophosphonic acidhydrochloride (3.70 g, 92%) as a solid.

Example 8

Monophosphonamidate 8: A mixture of monophosphonic acid 7 (0.22 g, 0.50mmol), L-alanine methyl ester hydrochloride (0.14 g, 1.00 mmol), andtriethylamine (0.21 mL, 1.50 mmol) in pyridine (3 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.39 g, 1.75 mmol) and triphenylphosphine (0.46 g, 1.75 mmol) inpyridine (2 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (7% MeOH/CH₂Cl₂) to givethe monophosphonamidate (97 mg, 39%, 1:1 diastereomeric mixture) as anoff-white foam.

Example 9

Monophosphonamidate 9: A mixture of montophosphonic acid 7 (0.88 g, 2.00mmol), D-alanine methyl ester hydrochloride (0.84 g, 6.00 mmol), andtriethylamine (0.84 mL, 6.00 mmol) in pyridine (8 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(1.56 g, 7.00 mmol) and triphenylphosphine (1.84 g, 7.00 mmol) inpyridine (8 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (7% MeOH/CH₂Cl₂) to givethe monophosphonamidate (0.40 g, 41%, 1:1 diastereomeric mixture) as anoff-white foam.

Example 10

Monophosphonamidate 10: A mixture of monophosphonic acid 7 (0.88 g, 2.00mmol), L-alanine tert-butyl ester hydrochloride (1.31 g, 6.00 mmol), andtriethylamine (0.84 mL, 6.00 mmol) in pyridine (8 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(1.54 g, 7.00 mmol) and triphenylphosphine (1.84 g, 7.00 mmol) inpyridine (8 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (7% MeOH/CH₂Cl₂) to givethe monophosphonamidate (0.38 g, 36%, 1:1 diastereomeric mixture) as alight orange foam.

Example 11

Monophosphonamidate 11: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine ethyl ester hydrochloride (94 mg, 0.60 mmol), phenol(0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol) in pyridine(1.0 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.47 g, 2.13 mmol) and triphenylphosphine(0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (7%MeOH/CH₂Cl₂) to give the monophosphonamidate (74 mg, 48%, 1:1diastereomeric mixture) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.61 (d,J=4.2 Hz, 1H), 7.26-7.08 (m, 5H), 4.23 (m, 2H), 4.13 (m, 2H), 4.09 (m,1H), 3.92-3.85 (m, 4H), 3.03 (broad, s, 1H), 1.30-1.26 (m, 3H), 1.24 (m,3H), 0.88 (m, 2H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ 21.94, 20.68.

Example 12

Monophosphonamidate 12: A mixture of phosphonic acid 6 (1.50 g, 4.56mmol), L-alamine n-propyl ester hydrochloride (1.59 g, 9.49 mmol),phenol (2.25 g, 22.80 mmol) and triethylamine (10.50 mL, 54.72 mmol) inpyridine (8.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (6.54 g, 31.92 mmol) andtriphenylphosphine (7.32 g, 31.92 mmol) in pyridine (8.0 mL) was addedto the above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (7% MeOH/CH₂Cl₂) to give themonophosphonamidate (0.43 g, 18%, Compound E, 1:1 diastereomericmixture) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.61 (d, J=5.1 Hz, 1H),7.27-7.09 (m, 5H), 4.27-4.20 (m, 2H), 4.16-4.00 (m, 3H), 3.93-3.82 (m,4H), 3.04 (broad, s, 1H), 1.63 (m, 2H), 1.30 (dd, 3H), 0.92 (m, 3H),0.89 (m, 2H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ 21.89, 20.66.

Example 13

Monophosphonamidate 13: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine isopropyl ester hydrochloride (0.10 g, 0.60 mmol),phenol (0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol) inpyridine (1.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.13 mmol) andtriphenylphosphine (0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (7% MeOH/CH₂Cl₂) to give themonophosphonamidate (87 mg, 55%, 1:1 diastereomeric mixture) as a yellowfoam: ¹H NMR (CDCl₃) δ 7.60 (d, J=2.1 Hz, 1H), 7.26-7.09 (m, 5H), 4.98(m, 1H), 4.23 (m, 2H), 4.06 (m, 1H), 3.91-3.83 (m, 4H), 3.04 (broad, s,1H), 1.29-1.21 (m, 9H), 0.89 (m, 2H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ21.85, 20.68.

Example 14

Monophosphonamidate 14: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine n-butyl ester hydrochloride (0.11 g, 0.60 mmol), phenol(0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol) in pyridine(1.0 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.47 g, 2.13 mmol) and triphenylphosphine(0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (7%MeOH/CH₂Cl₂) to give the monophosphonamidate (80 mg, 50%, 1:1diastereomeric mixture) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.61 (d,J=4.20 Hz, 1H), 7.27-7.08 (m, 5H); 5.93 (broad, s, 1H), 4.97 (broad, s,2H), 4.23 (m, 2H), 4.10-4.08 (m, 3H), 3.91-3.84 (m, 4H), 3.03 (broad, s,1H), 1.58 (m, 2H), 1.34-1.27 (m, 5H), 0.92-0.89 (m, 5H), 0.63 (m, 2H);³¹P NMR (CDCl₃) δ 21.94, 20.68.

Example 15

Monophosphonamidate 15: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine n-hexyl ester hydrochloride (0.13 g, 0.60 mmol), phenol(0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol) in pyridine(1.0 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.47 g, 2.13 mmol) and triphenylphosphine(0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the monophosphonamidate (0.10 g, 59%, 1:1 diastereomeric mixture)as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.59 (d, J=4.20 Hz, 1H),7.26-7.08 (m, 5H), 4.22 (m, 2H), 4.11 (m, 1H), 4.06 (m, 2H), 3.91-3.84(m, 4H), 3.01 (broad, s, 1H), 1.59 (m, 2H), 1.31-1.27 (m, 9H), 0.89 (m,3H), 0.86 (m, 2H), 0.62 (m, 2H); ³¹P NMR (CDCl₃) δ 21.94, 20.68.

Example 16

Monophosphonamidate 16: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine n-octanyl ester hydrochloride (0.15 g, 0.60 mmol),phenol (0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol) inpyridine (1.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.13 mmol) andtriphenylphosphine (0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphonamidate (0.13 g, 73%,1:1 diastereomeric mixture) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.59(d, J=4.2 Hz, 1H), 7.25-7.07 (m, 5H), 4.22 (m, 2H), 4.10 (m, 1H), 4.07(m, 2H), 3.90-3.84 (m, 4H), 3.02 (broad, s, 1H), 1.59 (m, 2H), 1.29-1.26(m, 113H), 0.88 (m, 3H), 0.85 (m, 2H), 0.60 (m, 2H); ³¹P NMR (CDCl₃) δ21.96, 20.69.

Example 17

Monophosphonamidate 17: A mixture of phosphonic acid 6 (70 mg, 0.21mmol), L-2-aminobutyric acid ethyl ester hydrochloride (72 mg, 0.42mmol), phenol (0.10 g, 1.05 mmol) and triethylamine (0.36 mL, 2.52 mmol)in pyridine (1.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.33 g, 1.47 mmol) andtriphenylphosphine (0.39 g, 1.47 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (7% MeOH/CH₂Cl₂) to give themonophosphonamidate (66 mg, 60%, 1:1 diastereomeric mixture) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.61 (d, J=7.2 Hz, 1H), 7.26-7.08 (m, 5H),5.91 (broad, s, 1H), 4.97 (broad, s, 2H), 4.22-4.12 (m, 4H), 4.01-3.81(m, 5H), 3.03 (broad, s, 1H), 1.71-1.60 (m, 2H), 1.24 (m, 3H), 0.89 (m,2H), 0.84-0.76 (m, 3H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ 22.15, 20.93.

Example 18

Monophosphonamidate 18: A mixture of phosphonic acid 6 (1.00 g, 3.05mmol), L-2-aminobutyric acid n-butyl ester hydrochloride (1.19 g, 6.09mmol), phenol (1.43 g, 15.23 mmol) and triethylamine (5.10 mL, 36.60mmol) in pyridine (5.0 mL) was heated to 60° C. for 5 min. A freshlyprepared bright yellow solution of aldrithiol (4.70 g, 21.32 mmol) andtriphenylphosphine (5.59 g, 21.32 mmol) in pyridine (5.0 mL) was addedto the above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (5% MeOH/CH₂Cl₂) to give themonophosphonamidate (0.7 g, 42%, Compound G, 1:1 diastereomeric mixture)as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d, J=6.60 Hz, 1H),7.27-7.04 (m, 5H), 5.89 (broad, s, 11H), 4.94 (broad, s, 2H), 4.22 (m,2H), 4.07-3.99 (m, 3H), 3.91-3.84 (m, 4H), 3.03 (broad, s, 1H),1.70-1.57 (m, 4H), 1.35 (m, 2H), 0.92-0.75 (m, 8H), 0.63 (m, 2H); ³¹PNMR (CDCl₃) δ 22.21, 20.95.

Example 19

Monophosphonamidate 19: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-2-aminobutyric acid n-octanyl ester hydrochloride (0.15 g, 0.60mmol), phenol (0.14 g, 1.52 mmol) and triethylamine (0.51 mL, 3.60 mmol)in pyridine (1.0 mL) was heated to 60° C. for 5 mm. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.13 mmol) andtriphenylphosphine (0.56 g, 2.13 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphonamidate (0.12 g, 64%,1:1 diastereomeric mixture) as a pale yellow foam: ¹H NMR (CDCl₃) δ 7.62(d, J=6.60 Hz, 1H), 7.25-7.08 (m, 5H), 4.24-4.21 (m 2H), 4.09-4.04 (m,2H), 4.00 (m, 1H), 3.91-3.83 (m, 4H), 3.01 (broad, s, 1H), 1.70-1.58 (m,4H), 1.27 (m, 10H), 0.89-0.76 (m, 8H), 0.62 (m, 2H); ³¹P NMR (CDCl₃) δ22.22, 20.92.

Example 20

Monophosphonamidate 20: A mixture of phosphonic acid 6 (1.0 g, 4.57mmol), L-phenylalanine ethyl ester hydrochloride (2.10 g, 9.14 mmol),phenol (2.15 g, 22.85 mmol) and triethylamine (7.64 mL, 54.84 mmol) inpyridine (8.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (7.05 g, 31.99 mmol) andtriphenylphosphine (8.39 g, 31.99 mmol) in pyridine (7.0 mL) was addedto the above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (5% MeOH/CH₂Cl₂) to give a pale yellowsolid 1.32 g containing about 10% impurity. The yellow solid (1.32 g,2.28 mmol) was dissolved in iPrOH (10 mL) and transferred to a hot iPrOH(30 mL) solution of fumaric acid (0.27 g, 2.28 mmol) and stirred at 80°C. for 30 min. The reaction mixture was gradually cooled to roomtemperature and the fumarate salt was collected at 0° C. The resultingfumarate salt was neutralized by partition from NaHCO₃ (2×) and EtOAc.The organic phase was washed with brine, H₂O, dried with Na₂SO₄,filtered, and concentrated. The product was dried under vacuum to givethe monophosphonamidate (0.70 g, 26%, Compound A, 1:1 diastereomericmixture) as a white foam: ¹H NMR (CDCl₃) δ 7.54 (d, J=2.4 Hz, 1H),7.27-6.98 (m, 10H), 4.35 (m, 1H), 4.16 (m, 2H), 4.08 (m, 2H), 3.84-3.61(m, 3H), 3.33 (m, 1H), 3.02 (broad, s, 1H), 2.95-2.87 (m, 2H), 1.17 (m,3H), 0.87 (m, 2H), 0.61 (m, 2H); ³¹P NMR (CDCl₃) δ 21.88, 21.07.

Example 21

Monophosphonamidate 21: A mixture of phosphonic acid 6 (70 mg, 0.21mmol), L-phenylamine n-butyl ester hydrochloride (0.11 g, 0.42 mmol),phenol (0.10 g, 1.05 mmol) and triethylamine (0.36 mL, 2.52 mmol) inpyridine (1.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.33 g, 1.47 mmol) andtriphenylphosphine (0.39 g, 1.47 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (7% MeOH/CH₂Cl₂) to give themonophosphonamidate (30 mg, 23%, 1:1 diastereomeric mixture) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.55 (d, J=2.7 Hz, 1H), 7.25-6.98 (m,10H), 4.36 (m, 1H), 4.17 (m, 2H), 4.02 (m, 2H), 3.83-3.35 (m, 4H), 3.02(broad, s, 1H), 2.94-2.86 (m, 2H), 1.52 (m, 2H), 1.29 (m, 2H), 0.90 (m,3H), 0.88 (m, 2H), 0.62 (m, 2H); ³¹P NMR (CDCl₃) δ 21.85, 21.05.

Example 22

Monophosphonamidate 22: A mixture of phosphonic acid 6 (70 mg, 0.21mmol), L-phenylalanine isobutyl ester hydrochloride (0.11 g, 0.42 mmol),phenol (0.10 g, 1.05 mmol) and triethylamine (0.36 mL, 2.52 mmol) inpyridine (1.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.33 g, 1.47 mmol) andtriphenylphosphine (0.39 g, 1.47 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (7% MeOH/CH₂Cl₂) to give themonophosphonamidate (65 mg, 50%, 1:1 diastereomeric mixture) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.56 (d, J=3.6 Hz, 1H), 7.26-6.98 (m,10H), 4.40 (m, 1H), 4.17 (m, 2H), 3.82 (m, 2H), 3.75-3.62 (m, 3H), 3.35(m, 1H), 3.04 (broad, s, 1H), 2.96-2.87 (m, 2H), 1.83 (m, 1H), 0.90 (m,2H), 0.86 (m, 6H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ 21.82, 21.03.

Example 23

Bisphosphonamidate 23: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine ethyl ester hydrochloride (0.28 g, 1.80 mmol), andtriethylamine (0.51 mL, 3.60 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.10 mmol) and triphenylphosphine (0.56 g, 2.10mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (80 mg, 50%,) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.63 (s, 1H), 5.88 (broad, s, 1H), 4.96(broad, s, 2H), 4.24-4.16 (m, 6H), 4.00 (m, 2H), 3.86 (m, 2H), 3.72 (m,2H), 3.01 (broad, s, 1H), 1.36 (m, 6H), 1.26 (m, 6H), 0.86 (m, 2H), 0.61(m, 2H); ³¹P NMR (CDCl₃) δ 20.63.

Example 24

Bisphosphonamidate 24: A mixture of phosphoric acid 6 (1.00 g, 3.05mmol), L-alamine n-propyl ester hydrochloride (3.06 g, 18.30 mmol), andtriethylamine (5.10 mL, 36.50 mmol) in pyridine (5.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (4.70 g, 21.32 mmol) and triphenylphosphine (5.59 g, 21.32mmol) in pyridine (5.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (1.13 g, 71%, Compound F) asa pale yellow foam: ¹H NMR (CDCl₃) δ 7.65 (s, 1H), 5.92 (broad, s, 1H),5.03 (broad, s, 2H), 4.24 (m, 2H), 4.10-4.02 (m, 6H), 3.87 (m, 2H), 3.73(m, 2H), 3.03 (broad, s, 1H), 1.65 (m, 4H), 1.37 (m, 6H), 0.93 (m, 6H),0.88 (m, 2H), 0.63 (m, 2H); ³¹P NMR (CDCl₃) δ 20.61.

Example 25

Bisphosphonamidate 25; A mixture of phosphonic acid 6 (0.60 g, 1.83mmol), L alanine isopropyl ester hydrochloride (1.84 g, 10.98 mmol), andtriethylamine (3.06 mL, 21.96 mmol) in pyridine (3.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (2.82 g, 12.80 mmol) and triphenylphosphine (3.36 g, 12.80mmol) in pyridine (3.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (0.53 g, 52%, Compound B) asa pale yellow foam: ¹H NMR (CDCl₃) δ 7.65 (s, 1H), 5.00 (m, 2H), 4.24(m, 2H), 3.97 (m, 2H), 3.87 (m, 2H), 3.71 (m, 2H), 3.01 (broad, s, 1H),1.34 (m, 6H), 1.23 (m, 12H), 0.86 (m, 2H), 0.62 (m, 2H); ³¹P NMR (CDCl₃)δ 20.59.

Example 26

Bisphosphonamidate 26: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine n-butyl ester hydrochloride (0.33 g, 1.82 mmol), andtriethylamine (0.51 mL, 3.60 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g 2.10 mmol) and triphenylphosphine (0.56 g, 2.10 mmol)in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (97 mg, 55%) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.63 (s, 1H), 4.24 (m, 2H), 4.09 (m, 4H),4.01 (m, 2H), 3.86 (m, 2H), 3.72 (m, 2H), 3.01 (broad, s, 1H), 1.61 (m,4H), 1.37 (m, 1014), 0.93 (m, 6H), 0.88 (m, 2H), 0.61 (m, 2H); ³¹P NMR(CDCl₃) δ 20.59.

Example 27

Bisphosphonamidate 27: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alamine n-hexyl ester hydrochloride (0.38 g, 1.80 mmol), andtriethylamine (0.51 mL, 3.60 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.10 mmol) and triphenylphosphine (0.56 g, 2.10mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphonamidate (0.13 g, 65%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.62 (s, 1H), 4.23 (m, 2H), 4.09 (m, 4H), 4.01 (m, 2H), 3.86(m, 2H), 3.72 (m, 2H), 2.99 (broad, s, 1H), 1.61 (m, 4H), 1.36-1.29 (m,18H), 0.88 (m, 6H), 0.84 (m, 2H), 0.60 (m, 2H); ³¹P NMR (CDCl₃) δ 20.61.

Example 28

Bisphosphonamidate 28: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-alanine n-octanyl ester hydrochloride (0.43 g, 1.80 mmol), andtriethylamine (0.51 mL, 3.60 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.10 mmol) and triphenylphosphine (0.56 g, 2.10mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphonamidate (0.13 g, 61%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.61 (s, 1H), 4.21 (m, 2H), 4.07-4.00 (m, 6H), 3.84-3.70 (m,4H), 2.98 (broad, s, 1H), 1.60 (m, 4H), 1.34 (m, 6H), 1.27 (m, 20H),0.87 (m, 6H), 0.83 (m, 2H), 0.58 (m, 2H); ³¹P NMR (CDCl₃) δ 20.63.

Example 29

Bisphosphonamidate 29: A mixture of phosphoric acid 6 (0.70 g, 2.13mmol), L-2-aminobutyric acid ethyl ester hydrochloride (2.15 g, 12.80mmol), and triethylamine (3.57 mL, 25.56 mmol) in pyridine (3.0 mL) washeated to 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (3.29 g, 14.91 mmol) and triphenylphosphine (3.92 g, 14.91mmol) in pyridine (3.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (0.71 g, 60%, Compound D) asa pale yellow foam: ¹H NMR (CDCl₃) δ 7.64 (s, 1H), 4.24 (m, 2H), 4.16(m, 4H), 3.89-3.87 (m, 4H), 3.72 (d, J=9.0 Hz, 2H), 3.01 (road, s, 1H),1.78-1.64 (m, 4H), 1.26 (m, 6H), 0.91 (m, 6H), 0.87 (m, 2H), 0.61 (m,2H); ³¹P NMR (CDCl₃) δ 21.23.

Example 30

Bisphosphonamidate 30: A mixture of phosphonic acid 6 (0.70 g, 21.32mmol), L-2-aminobutyric acid n-butyl ester hydrochloride (2.50 g, 12.80mmol), and triethylamine (3.57 mL, 25.56 mmol) in pyridine (3.0 mL) washeated to 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (3.29 g, 14.91 mmol) and triphenylphosphine (3.92 g, 14.91mmol) in pyridine (3.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (0.40 g, 31%, Compound C) asa pale yellow foam: ¹H NMR (CDCl₃) δ 7.64 (s, 11H), 4.24 (m, 2H), 4.11(m, 4H), 3.91 (m, 2H), 3.87 (m, 2H), 3.71 (d, J=9.0 Hz, 2H), 3.03(broad, s, 1H), 1.79-1.64 (m, 4H), 1.60 (m, 4H), 1.37 (m, 4H), 0.94 (m,6H), 0.90 (m, 6H), 0.86 (m, 2H), 0.62 (m, 2H); ³¹P NMR (CDCl₃) δ 21.25.

Example 31

Bisphosphonamidate 31: A mixture of phosphonic acid 6 (0.10 g, 0.30mmol), L-2-aminobutyric acid n-octanyl ester hydrochloride (0.33 g, 1.82mmol), and triethylamine (0.51 mL, 3.60 mmol) in pyridine (1.0 mL) washeated to 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.10 mmol) and triphenylphosphine (0.56 g, 2.10mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphonamidate (0.12 g, 55%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.64 (s, 1H), 4.24 (m, 2H), 4.13-4.05 (m, 4H), 3.91 (m, 2H),3.87-3.72 (m, 4H), 3.01 (broad, s, 1H), 1.78-1.65 (m, 4H), 1.61-1.29 (m,24H), 0.91 (m, 6H), 0.89 (m, 6H), 0.86 (m, 2H), 0.62 (m, 2H); ³¹P NMR(CDCl₃) δ 21.20.

Example 32

Bisphosphonamidate 32: A mixture of phosphonic acid 6 (0.60 g, 1.82mmol), L-phenylalanine ethyl ester hydrochloride (2.51 g, 10.96 mmol),and triethylamine (3.06 mL, 21.84 mmol) in pyridine (3.0 mL) was heatedto 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (2.82 g, 1.2.74 mmol) and triphenylphosphine (3.36 g, 12.74mmol) in pyridine (3.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphonamidate (0.53 g, 43%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.48 (s, 1H), 7.22-7.06 (m, 10H), 4.20 (m, 1H), 4.12 (m, 4H),4.09 (m, 2H), 4.04 (m, 1H), 3.63 (m, 2H), 3.33-3.21 (m, 2H), 3.04-2.78(m, 5H), 1.20 (m, 6H), 0.83 (m, 2H), 0.58 (m, 2H); ³¹P NMR (CDCl₃) δ20.38.

Example 33

Bisphosphonamidate 33. A mixture of phosphonic acid 6 (70 mg, 0.21mmol), L-phenylalanine n-butyl ester hydrochloride (0.33 g, 1.26 mmol),and triethylamine (0.36 mL, 2.52 mmol) in pyridine (1.0 mL) was heatedto 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.33 g, 1.47 mmol) and triphenylphosphine (0.39 g, 1.47mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (0.11 g, 70%) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.51 (s, 1H), 7.23-7.06 (m, 10H), 4.23 (m,1H), 4.11-4.05 (m, 7H), 3.65 (m, 2H), 3.35-3.23 (m, 2H), 3.01 (m, 1H),3.04-2.78 (m, 4H), 1.57 (m, 4H), 1.33 (m, 4H), 0.92 (m, 6H), 0.86 (m,2H), 0.61 (m, 2H); ³¹P NMR (CDCl₃) δ 20.35.

Example 34

Bisphosphonamidate 34: A mixture of phosphonic acid 6 (70 mg, 0.21mmol), L-phenylalanine isobutyl ester hydrochloride (0.33 g, 1.26 mmol),and triethylamine (0.36 mL, 2.52 mmol) in pyridine (1.0 mL) was heatedto 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.33 g, 1.47 mmol) and triphenylphosphine (0.39 g, 1.47mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (10%MeOH/CH₂Cl₂) to give the bisphosphonamidate (78 mg, 50%) as a paleyellow foam: ¹H NMR (CDCl₃) δ 7.52 (s, 1H), 7.24-7.07 (m, 10H), 4.26 (m,11H), 4.11 (m, 2H), 4.01 (m, 1H), 3.85 (m, 4H), 3.66 (m, 2H), 3.35-3.25(m, 2H), 3.07-2.85 (m, 3H), 2.97-2.79 (m, 2H), 1.89 (m, 2H), 0.90 (m,121H), 0.89 (m, 2H), 0.62 (m, 2H); ³¹P NMR (CDCl₃) δ 20.31.

Example 35

BisPOC of cPrPMEDAP 35: A mixture of phosphonic acid 6 (0.20 g, 0.61mmol) and triethylamine (0.42 mL, 3.01 mmol) in 1-methyl-2-pyrrolidinone(2.0 mL) was heated to 60° C. for 30 min. POCCI (0.45 g, 2.92 mmol) wasadded. The reaction mixture was stirred at 60° C. for 3 h, cooled toroom temperature, and concentrated. The product was partitioned betweenEtOAc and saturated NaHCO₃. The organic phase was washed with brine,dried with Na₂SO₄, filtered, and evaporated under reduced pressure. Thecrude product was purified by chromatography on ISCO (2-propanol/CH₂Cl₂)to give the bisPOC of cPrPMEDAP (0.13 g, 39%) as a solid: ¹H NMR (CDCl₃)δ 7.58 (s, 1H), 5.66 (m, 4H), 4.92 (m, 2H), 4.22 (m, 2H), 3.90-3.88 (m,4H), 3.01 (broad, s, 1H), 1.81 (m, 12H), 0.86 (m, 2H), 0.62 (m, 2H); ³¹PNMR (CDCl₃) δ 20.93.

Examples 36 to 38 Refer to Scheme 10 Example 36

Bisphosphonamidate 37: A mixture of phosphonic acid 36 (0.32 g, 1.00mmol), L-alanine butyl ester hydrochloride (0.47 g, 2.60 mmol), andtriethylamine (0.27 g, 2.60 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.77 g, 3.50 mmol) and triphenylphosphite (0.92 g, 3.50 mmol) inpyridine (2.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (10% MeOH/CH₂Cl₂) togive the bisphosphonamidate (0.43 g, 75%) as a pale yellow foam.

Example 37

Monophosphonic Acid 38: A mixture of diacid 36 (1.30 g, 4.10 mmol) andDMF (0.1 mL) in sulfolane (35 mL) was heated to 70° C. Thionylchloride(0.54 mL, 7.38 mmol) was added dropwise over a period of 1 h. Thetemperature was increased to 90° C. and TMSOPh (0.75 g, 4.51 mmol) wasadded and stirred for 1 h. The reaction mixture was cooled to roomtemperature overnight. The reaction mixture was added dropwise towell-stirred, ice-cold acetone (100 mL). The product was precipitatedout. The solid was filtered and dissolved in MeOH (40 mL) and pH wasadjusted to 3 with 45% KOH. Solid was collected by filtration. Theproduct was further purified by dissolving in MeOH, adjusting pH to 6with 45% KOH, and crystallizing from ice-cold acetone to give themonophosphonic acid (0.20 g, 12%) as an off-white solid.

Example 38

Monophosphonamidate 39: A mixture of monophosphonic acid 38 (0.20 g,0.50 mmol), L-alanine isopropyl ester hydrochloride (0.17 g, 1.00 mmol)and triethylamine (0.10 g, 1.00 mmol) in pyridine (2.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.39 g, 1.75 mmol) and triphenylphosphine (0.46 g, 1.75mmol) in pyridine (2.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (7%MeOH/CH₂Cl₂) to give the monophosphonamidate (0.14 g, 54%, 1:1diastereomeric mixture) as a pale yellow foam.

Example 39 Refers to Scheme 11 Example 39

Bisphosphonamidate 41: A mixture of phosphonic acid 40 (0.36 g, 1.00mmol), L-alanine n-butyl ester hydrochloride (0.47 g, 2.60 mmol), andtriethylamine (0.27 g, 2.60 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.77 g, 3.50 mmol) and triphenylphosphine (0.92 g, 3.50 mmol) inpyridine (2.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (10% MeOH/CH₂Cl₂) togive the bisphosphonamidate (0.32 g, 35%) as a pale yellow foam.

Examples 40 to 56 Refer to Schemes 12 to 16 Example 40

Bisphosphonamidate 43: A mixture of phosphonic acid 42 (0.37 g, 1.00mmol), L-alanine n-butyl ester hydrochloride (0.47 g, 2.60 mmol), andtriethylamine (0.27 g, 2.60 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.77 g, 3.50 mmol) and triphenylphosphine (0.92 g, 3.50 mmol) inpyridine (2.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (10% MeOH/CH₂Cl₂) togive the bisphosphonamidate (0.53 g, 85%) as a pale yellow foam.

Example 41

Bisphosphonamidate 45: A mixture of phosphonic acid 44 (0.55 g, 2.00mmol), L-alanine butyl ester hydrochloride (0.94 g, 5.20 mmol), andtriethylamine (0.54 g, 5.20 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(1.54 g, 7.00 mmol) and triphenylphosphine (1.84 g, 7.00 mmol) inpyridine (5.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (10% MeOH/CH₂Cl₂) togive the bisphosphonamidate (0.48 g, 45%) as a pale yellow foam.

Example 42

Monophosphonic Acid 46: A mixture of diacid 44 (10.00 g, 36.30 mmol) andDMF (0.2 mL) in sulfolane (50 mL) was heated to 70° C. Thionylchloride(4.72 mL, 64.70 mmol) was added dropwise over a period of 1 h. Thetemperature was increased to 90° C. and TMSOPh (6.65 g, 40.00 mmol) wasadded and stirred for 1 h. The reaction mixture was cooled to roomtemperature overnight. The reaction mixture was added dropwise towell-stirred, ice-cold acetone (100 mL). The product was precipitatedout. The solid was filtered and dissolved in MeOH (40 mL) and pH wasadjusted to 3 with 45% KOH. Solid was collected by filtration and driedunder vacuum to give the monophosphonic acid (12.40 g, 97%) as a solid.

Example 43

Monophosphonamidate 47: A mixture of monophosphonic acid 46 (1.00 g,2.86 mmol), L-alanine methyl ester hydrochloride (0.80 g, 5.73 mmol) andtriethylamine (0.58 g, 5.73 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 mL. A freshly prepared bright yellow solution of aldrithiol(2.21 g, 10.00 mmol) and triphenylphosphine (2.63 g, 10.00 mmol) inpyridine (5.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (7% MeOH/CH₂Cl₂) to givethe monophosphonamidate (0.80 g, 64%, 1:1 diastereomeric mixture) as apale yellow oil.

Example 44

Monophosphonamidate 48: A mixture of monophosphonic acid 46 (0.35 g,1.00 mmol), L-alanine isopropyl ester hydrochloride (0.34 g, 2.00 mmol)and triethylamine (0.20 g, 2.00 mmol) in pyridine (2.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.77 g, 3.50 mmol) and triphenylphosphine (0.92 g, 3.50mmol) in pyridine (2.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (7%MeOH/CH₂Cl₂) to give the monophosphonamidate containing some impurity.The resulting compound was treated with fumaric acid (77 mg) in hotCH₃CN (10 mL) and cooled to room temperature. The product wasprecipitated out and dried under vacuum to give the fumarate salt ofmonophosphonamidate (0.13 g, 22%, 1:1 diastereomeric mixture) as asolid.

Example 45

Benzyl Ether of PMEG 50: A mixture of diacid 49 (0.62 g, 2.00 mmol) andbenzyl alcohol (10 mL) was cooled to 0° C. with stirring. Sodium hydride(0.24 g, 10.00 mmol) was added portion wise and the reaction mixture washeated to 100° C. over 1 h. Additional benzyl alcohol (20 mL) and sodiumhydride (0.12 g, 5.00 mmol) were added. The reaction was stirred at 140°C. for 1 h and cooled to room temperature. The volatiles were evaporatedunder reduced pressure, water (50 mL) was added, and the pH was adjustedto 11 with NaOH. The product was partitioned between toluene (3×) andH₂O. The aqueous phase was acidified with HCl to pH 3 and kept at 0° C.overnight. The product was collected and dried under vacuum to give thebenzyl ether (0.18 g, 22%) as a tan solid.

Example 46

Monophosphonamidate 51: A mixture of phosphonic acid 50 (0.13 g, 0.34mmol), L-alanine isopropyl ester hydrochloride (0.11 g, 0.68 mmol),phenol (0.16 g, 1.69 mmol) and triethylamine (0.28 mL, 2.03 mmol) inpyridine (2.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.52 g, 2.37 mol) andtriphenylphosphine (0.62 g, 2.37 mmol) in pyridine (2.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (5% MeOH/CH₂Cl₂) to give themonophosphonamidate (50 mg, 26%, 1:1 diastereomeric mixture) as a thickoil.

Example 47

Monophosphonamidate 52: A mixture of monophosphonamidate 51 (50 mg, 0.09mmol) and Pd(OH)₂/C (50 mg) in iPrOH (3 mL) was stirred at roomtemperature under 1 atm of H₂ (balloon) overnight. The reaction mixturewas filtered through a plug of celite and the solvent was removed onrotavap under reduced pressure. The crude product was purified by columnchromatography on silica gel (5-15% MeOH/CHCl₃) to give themonophosphonamidate (40 mg, 95%, 1:1 diastereomeric mixture) as anoff-white foam.

Example 48

Bisphosphonamidate 54: A mixture of phosphonic acid 53 (0.10 g, 0.35mmol), L-alanine butyl ester hydrochloride (0.38 g, 2.10 mmol), andtriethylamine (0.58 mL, 4.20 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.53 g, 2.45 mmol) and triphenylphosphine (0.64 g, 2.45mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnights cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by column chromatography on silica gel (15%MeOH/CH₂Cl₂) to give the bisphosphonamidate (25 mg, 13%) as a paleyellow foam: ¹H NMR (CD₃OD) δ 7.82 (s, 1H), 4.26 (m, 2H), 4.11 (m, 4H),3.94 (m, 2H), 3.88 (m, 2H), 3.78 (m, 2H), 1.61 (m, 4H), 1.39 (m, 4H),1.34 (m, 6H), 0.95 (m, 6H); ³¹P NMR (CDCl₃) δ 23.39.

Example 49

Diisopropyl Phosphonate 55: A mixture of 4 (3.00 g, 7.66 mmol) and 10%Pd/C (0.60 g) in MeOH (30 mL) was stirred at room temperature under 1atm of Hz (balloon) overnight. The reaction mixture was filtered througha plug of celite and the solvent was removed on rotavap. The crudeproduct was purified by column chromatography on silica gel (5%MeOH/CHCl₃) to give the diisopropyl phosphonate (2.08 g, 76%) as a thickoil which was solidified upon standing: ¹H NMR (CDCl₃) δ 8.72 (s, 1H),7.94 (s, 1H), 4.73 (m, 2H), 4.33 (m, 2H), 3.97 (m, 2H), 3.73 (d, J=8.1Hz, 2H), 1.31 (m, 12H); ³¹P NMR (CDCl₃) δ 18.47.

Example 50

Phosphonic Acid 56 Diisopropyl phosphonate 55 (0.10 g, 0.28 mmol) wasdissolved in CH₃CN (1.5 mL) and cooled to 0° C. Bromotrimethylsilane(0.18 mL, 1.40 mmol) was added. The reaction mixture was stirred at 0°C. for 2 h and warmed to room temperature overnight. DMF (0.5 mL) wasadded to form a solution and stirred for 2 h. MeOH was added and stirredfor 2 h. Volatiles were evaporated under reduced pressure. The remainingDMF solution was added slowly to ice-cold CH₃CN and the productprecipitated out. The solid was collected and dried under vacuum to givethe phosphonic acid (74 mg, 95%) as a white solid.

Example 51

Bisphosphonamidate 57: A mixture of phosphonic acid 56 (23 mg, 0.08mmol), L-alanine n-butyl ester hydrochloride (91 mg, 0.50 mmol), andtriethylamine (0.14 mL, 0.96 mmol) in pyridine (0.5 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.11 g, 0.56 mmol) and triphenylphosphine (0.12 g, 0.56mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphonamidate (17 mg, 38%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 8.65 (s, 1H), 7.94 (s, 1H), 5.20 (s, broad, 2H), 4.35 (m, 2H),4.20-3.92 (m, 6H), 3.89 (m, 2H), 3.72 (m, 2H), 3.42-3.19 (m, 2H), 1.61(m, 4H), 1.32 (m, 8H), 0.96 (m, 6H); ³¹P NMR (CDCl₃) δ 20.70.

Example 52

Monophosphonamidate 58: A mixture of phosphonic acid 56 (20 mg, 0.07mmol), L-phenylalanine ethyl ester hydrochloride (33 mg, 0.14 mmol),phenol (33 mg, 0.35 mmol) and triethylamine (0.12 mL, 0.84 mmol) inpyridine (0.5 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.11 g, 0.56 mmol) andtriphenylphosphine (0.12 g, 0.56 mmol) in pyridine (0.5 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphonamidate (13 mg, 34%,1:1 diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 8.69(d, J=15.0 Hz, 1H), 7.84 (d, J=4.2 Hz, 1H), 7.25-6.97 (m, 10H), 4.35 (m,1H), 4.23 (m, 2H), 4.08 (m, 2H), 3.85 (m, 1H), 3.72 (m, 1H), 3.73-3.62(m, 1H), 3.38 (m, 1H), 2.95-2.86 (m, 2H), 1.17 (m, 3H); ³¹P NMR (CDCl₃)δ 21.67, 20.84.

Example 53

Diisopropyl Phosphonate 59: A mixture of compound 4 (1.00 g, 2.56 mmol)and allylamine (3 mL) in CH₃CN (3.0 mL) was placed in a scintillationvial and heated to 65° C. for 5 h. The reaction mixture was cooled toroom temperature and concentrated under reduced pressure. The productwas partitioned between EtOAc and brine, dried with Na₂SO₄, filtered,and concentrated. The product was dissolved in minimal CH₃CN and H₂O wasadded and lyophilized to give the diisopropyl phosphonate (1.00 g, 95%).

Example 54

Phosphonic Acid 60: Diisopropyl phosphonate 59 (1.00 g, 2.43 mmol) wasdissolved in CH₃CN (1.5 mL) and cooled to 0° C. Bromotrimethylsilane(0.31 mL, 12.15 mmol) was added. The reaction mixture was stirred at 0°C. for 2 h and warmed to room temperature overnight. DMF (0.5 mL) wasadded to form a solution and stirred for 2 h. MeOH was added and stirredfor 2 h. Volatiles were evaporated under reduced pressure. The remainingDMF solution was added slowly to ice-cold CH₃CN and the productprecipitated out. The solid was collected and dried under vacuum to givethe phosphonic acid (0.48 g, 60%) as a white solid.

Example 55

Monophosphonamidate 61 and Bisphosphonamidate 62: A mixture of diacid 60(0.40 g, 1.20 mmol), L-alanine isopropyl ester hydrochloride (0.49 g,2.40 mmol), phenol (0.68 g, 7.20 mmol), and triethylamine (1.0 mL, 7.20mmol) in pyridine (3.0 mL) was heated to 60° C. for 5 min. A freshlyprepared bright yellow solution of aldrithiol (1.84 g, 8.40 mmol) andtriphenylphosphine (2.20 g, 8.40 mmol) in pyridine (3.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by columnchromatography on silica gel (5-10% MeOH/CH₂Cl₂) to givemonophosphonamidate 61 (0.52 g, 37%, 1:1 diastereomeric mixture) andbisphosphonamidate 62 (0.13 g, 20%).

Example 56

Bisphosphonamidate 63: A mixture of phosphonic acid 60 (0.33 g, 1.00mmol), L-alanine butyl ester hydrochloride (0.47 g, 2.60 mmol), andtriethylamine (0.27 g, 2.60 mmol) in pyridine (5.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.77 g, 3.50 mmol) and triphenylphosphine (0.92 g, 3.50 mmol) inpyridine (2.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel (10% MeOH/CH₂Cl₂) togive the bisphosphonamidate (0.32 g, 55%) as a pale yellow foam.

Example 57 Relates to Scheme 17 Example 57

BisPOC of 6-allylPMEDAP 64: A mixture of phosphonic acid 60 (0.20 g,0.61 mmol) and triethylamine (0.42 mL, 3.01 mmol) in1-methyl-2-pyrrolidinone (2.0 mL) was heated to 60° C. for 30 min. POCCl(0.45 g, 2.92 mmol) was added. The reaction mixture was stirred at 60°C. for 3 h, cooled to room temperature, and concentrated. The productwas partitioned between EtOAc and saturated NaHCO₃. The organic phasewas washed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the bisPOC of 6-allylPMEDAP 64 (0.11 g,32%, GS 192727) as a solid: ¹H NMR (CDCl₃) δ 7.60 (s, 1H), 6.00 (m, 1H),5.66 (m, 4H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.92 (m, 2H), 4.80 (s, 2H),4.22 (m, 4H), 3.95 (m, 4H), 1.35 (m, 12H); ³¹P NMR (CDCl₃) δ 20.94.

Examples 58 to 61 Relate to Scheme 18 Example 58

Bisphosphoamidate 65: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-alanine ethyl ester hydrochloride (0.1 g, 0.65 mmol), andtriethylamine (0.2 mL, 1.43 mmol) in pyridine (0.5 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.16 g, 0.74 mmol) and triphenylphosphine (0.20 g, 0.75 mmol) inpyridine (0.5 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by chromatography on ISCO) (2-propanol/CH₂Cl₂) to give thebisphosphoamidate (23 mg, 41%) as a pale yellow foam: ¹H NMR (CDCl₃) δ7.70 (s, 1H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.30 (m, 4H),4.20-4.00 (m, 6H), 3.89 (m, 2H), 3.72 (m, 2H), 3.42 (m, 1H), 3.22 (m,1H), 1.45-1.25 (m, 121H); ³¹P NMR (CDCl₃) δ 20.77.

Example 59

Bisphosphoamidate 66: A mixture of phosphonic acid 60 (0.10 g, 0.30mmol), L-alanine cyclobutyl ester hydrochloride (0.33 g, 0.91 mmol), andtriethylamine (0.50 mL, 3.59 mmol) in pyridine (2.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.12 mmol) and triphenylphosphine (0.56 g, 2.14mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphoamidate (45 mg, 26%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.60 (s, 1H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.18 (dd, 1H), 5.00(m, 2H), 4.78 (s, 2H), 4.30 (m, 4H), 4.00 (m, 2H), 3.89 (m, 2H), 3.72(m, 2H), 3.38 (m, 1H), 3.19 (m, 1H), 2.38 (m, 4H), 2.10 (m, 4H),1.85-1.60 (m, 4H), 1.45 (m, 6H); ³¹P NMR (CDCl₃) δ 20.61.

Example 60

Bisphosphoamidate 67: A mixture of phosphonic acid 60 (0.10 g, 0.30mmol), L-alanine n-hexyl ester hydrochloride (0.25 g, 1.21 mmol), andtriethylamine (0.7 mL, 5.02 mmol) in pyridine (2.0 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.47 g, 2.12 mmol) and triphenylphosphine (0.56 g, 2.14 mmol) inpyridine (1.0 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brine, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by chromatography on ISCO (2-propanol/CH₂Cl₂) to give thebisphosphoamidate (80 mg, 41%) as a pale yellow foam: ¹H NMR (CDCl₃) δ7.65 (s, 1H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.80 (s, 2H),4.25 (m, 4H), 4.20-4.00 (m, 6H), 3.85 (m, 2H), 3.72 (m, 2H), 3.42 (m,1H), 3.20 (m, 1H), 1.70 (m, 4H), 1.32 (m, 18H), 0.96 (m, 6H); ³¹P NMR(CDCl₃) δ 20.61.

Example 61

Bisphosphoamidate 68: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-2-aminobutyric acid n-butyl ester hydrochloride (0.13 g, 0.64mmol), and triethylamine (0.2 mL, 1.43 mmol) in pyridine (0.5 mL) washeated to 60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.16 g, 0.74 mmol) and triphenylphosphine (0.20 g, 0.75mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphoamidate (32 mg, 49%) as a pale yellow foam: ¹HNMR(CDCl₃) δ 7.68 (s, 1H), 6.00 (m, 1H), 5.70 (s, broad, 1H), 5.30 (dd,1H), 5.18 (dd, 1H), 4.80 (s, 2H), 4.25 (m, 4H), 4.20-4.05 (m, 6H), 3.89(m, 2H), 3.72 (m, 2H), 3.35 (m, 1H), 3.15 (m, 1H), 1.86-1.60 (m, 8H),1.40 (m, 4H), 0.96 (m, 12H); ³¹P NMR (CDCl₃) δ 21.25.

Examples 62 to 71 Related to Scheme 19 Example 62

Bisphosphoamidate 69: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-phenylamine ethyl ester hydrochloride (0.15 g, 0.65 mmol), andtriethylamine (0.2 mL, 1.43 mmol) in pyridine (0.5 mL) was heated to 60°C. for 5 min. A freshly prepared bright yellow solution of aldrithiol(0.16 g, 0.74 mmol) and triphenylphosphine (0.20 g, 0.75 mmol) inpyridine (0.5 mL) was added to the above reaction mixture. The reactionwas stirred at 60° C. overnight, cooled to room temperature, andconcentrated. The product was partitioned between EtOAc and saturatedNaHCO₃. The organic phase was washed with brie, dried with Na₂SO₄,filtered, and evaporated under reduced pressure. The crude product waspurified by chromatography on ISCO (2-propanol/CH₂Cl₂) to give thebisphosphoamidate (28 mg, 39%) as a pale yellow foam: ¹H NMR (CDCl₃) δ7.58 (s, 1H), 7.28-7.03 (m, 10H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd,1H), 4.25-4.00 (m, 8H), 3.65 (m, 2H), 3.42-3.19 (m, 2H), 3.15-2.77 (m,6H), 1.23 (m, 6H); ³¹P NMR (CDCl₃) δ 20.34.

Example 63

Bisphosphoamidate 70: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-phenylalanine n-butyl ester hydrochloride (0.15 g, 0.58 mmol),and triethylamine (0.2 mL, 1.43 mmol) in pyridine (0.5 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.16 g, 0.74 mmol) and triphenylphosphine (0.20 g, 0.75mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphoamidate (49 mg, 63%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.58 (s, 1H), 7.28-7.03 (m, 10H), 6.00 (m, 1H), 5.70 (s,broad, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.78 (s, 2H), 4.25-4.03 (m,8H), 3.65 (m, 2H), 3.42-3.19 (m, 2H), 3.17-2.78 (m, 6H), 1.61 (m, 4H),1.32 (m, 4H), 0.96 (m, 6H); ³¹P NMR (CDCl₃) δ 20.35.

Example 64

Bisphosphoamidate 71: A mixture of phosphoric acid 60 (0.10 g, 0.30mmol), L-phenylalanine isobutyl ester hydrochloride (0.31 g, 1.20 mmol),and triethylamine (0.7 mL, 5.02 mmol) in pyridine (2.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.44 g, 2.00 mmol) and triphenylphosphine (0.53 g, 2.00mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphoamidate (94 mg, 42%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.55 (s, 1H), 7.27-7.03 (m, 10H), 6.00 (m, 1H), 5.70 (s,broad, 1H), 5.25 (dd, 1H), 5.17 (dd, 1H), 4.78 (s, 2H), 4.25-4.08 (m,4H), 3.87 (m, 4H), 3.65 (m, 2H), 3.42-3.19 (m, 2H), 3.17-2.78 (m, 6H),1.97 (m, 2H), 0.96 (m, 12H); ³¹P NMR (CDCl₃) δ 20.31.

Example 65

Monophosphoamidate 72: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-alanine ethyl ester hydrochloride (32 mg, 0.20 mmol), phenol(50 mg, 0.53 mmol) and triethylamine (0.2 mL, 1.43 mmol) in pyridine(0.5 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.16 g, 0.74 mmol) and triphenylphosphine(0.20 g, 0.75 mmol) in pyridine (0.5 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the monophosphoamidate (12 mg, 22%, 1:1 diastereomeric mixture) asan off-white foam: ¹H NMR (CDCl₃) δ 7.62 (d, 1H), 7.30-7.04 (m, 5H),6.00 (m, 1H), 5.30 (dd, 1H), 5.18 (dd, 1H), 4.30-4.05 (m, 7H), 3.90-3.80(m, 4H), 1.23 (m, 6H); ³¹P NMR (CDCl₃) δ 21.89, 20.65.

Example 66

Monophosphoamidate 73: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-alanine n-butyl ester hydrochloride (39 mg, 0.21 mmol), phenol(50 mg, 0.53 mmol) and triethylamine (0.2 mL, 1.43 mmol) in pyridine(0.5 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.16 g, 0.74 mmol) and triphenylphosphine(0.20 g, 0.75 mmol) in pyridine (0.5 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the monophosphoamidate (16 mg, 28%, 1:1 diastereomeric mixture) asan off-white foam: ¹H NMR (CDCl₃) δ 7.61 (d, 1H), 7.32-7.06 (m, 5H),6.00 (m, 1H), 5.80 (s, broad, 1H), 5.30 (dd, 1H), 5.20 (dd, 1H), 4.80(m, 2H), 4.30-4.05 (m, 7H), 3.90-3.80 (m, 4H), 3.90-3.60 (m, 2H), 1.60(m, 2H), 1.32 (m, 5H), 0.96 (m, 3H); ³¹P NMR (CDCl₃) δ 21.96, 20.70.

Example 67

Monophosphoamidate 74: A mixture of phosphonic acid 60 (0.10 g, 0.30mmol), L-alanine cyclobutyl ester hydrochloride (0.11 g, 0.61 mmol),phenol (0.13 g, 1.39 mmol) and triethylamine (0.5 mL, 3.59 mmol) inpyridine (2.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.12 mmol) andtriphenylphosphine (0.56 g, 2.14 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphoamidate (28 mg, 17%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d,1H), 7.25-7.03 (m, 5H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.18 (dd, 1H), 5.00(m, 2H), 4.79 (d, 2H), 4.28-4.05 (m, 4H), 3.90 (m, 4H), 3.70 (m, 1H),3.57 (m, 1H), 2.30 (m, 2H), 2.00-1.60 (m, 4H), 1.25 (m, 3H); ³¹P NMR(CDCl₃) δ 21.91, 20.64.

Example 68

Monophosphoamidate 75: A mixture of phosphonic acid 60 (0.10 g, 0.30mmol), L-alanine n-hexyl ester hydrochloride (0.13 g, 0.61 mmol), phenol(0.14 g, 1.52 mmol) and triethylamine (0.7 mL, 5.02 mmol) in pyridine(2.0 mL) was heated to 60° C. for 5 min. A freshly prepared brightyellow solution of aldrithiol (0.47 g, 2.12 mmol) and triphenylphosphine(0.56 g, 2.14 mmol) in pyridine (1.0 mL) was added to the above reactionmixture. The reaction was stirred at 60° C. overnight, cooled to roomtemperature, and concentrated. The product was partitioned between EtOAcand saturated NaHCO₃. The organic phase was washed with brine, driedwith Na₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on. ISCO (2-propanol/CH₂Cl₂) togive the monophosphoamidate (28 mg, 16%, 1:1 diastereomeric mixture) asan off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d, 1H), 7.25-7.03 (m, 5H),6.00 (m, 1H), 5.85 (s, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.78 (d, 2H),4.35-4.05 (m, 7H), 3.90 (m, 4H), 3.70 (m, 1H), 1.60 (m, 2H), 1.30 (m,9H), 0.96 (m, 3H); ³¹P NMR (CDCl₃) δ 21.97, 20.69.

Examples 69 to 72 Relate to Scheme 21 Example 69

Monophosphoamidate 76. A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-2-aminobutyric acid n-butyl ester hydrochloride (42 mg, 0.21mmol), phenol (50 mg, 0.53 mmol) and triethylamine (0.2 mL, 1.43 mmol)in pyridine (0.5 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.16 g, 0.74 mmol) andtriphenylphosphine (0.20 g, 0.75 mmol) in pyridine (0.5 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphoamidate (17 mg, 29%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d,1H), 7.25-7.03 (m, 5H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H), 4.78(d, 2H), 4.30-4.03 (m, 7H), 3.95-3.80 (m, 4H), 3.62 (m, 1H), 3.40 (m,1H), 1.80-1.60 (m, 4H), 1.38 (m, 2H), 0.98-0.75 (m, 6H); ³¹P NMR (CDCl₃)δ 22.26, 20.95.

Example 70

Monophosphoamidate 77: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-phenylalanine ethyl ester hydrochloride (48 mg, 0.21 mmol),phenol (50 mg, 0.53 mmol) and triethylamine (0.2 mL, 1.43 mmol) inpyridine (0.5 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.16 g, 0.74 mmol) andtriphenylphosphine (0.20 g, 0.75 mmol) in pyridine (0.5 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphoamidate (14 mg, 23%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d,1H), 7.25-7.03 (m, 10H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H),4.80 (m, 2H), 4.40-4.08 (m, 7H), 3.85-3.65 (m, 4H), 3.38-3.25 (m, 2H),2.95-2.86 (m, 2H), 1.20 (m, 3H); ³¹P NMR (CDCl₃) δ 21.86, 21.06.

Example 71

Monophosphoamidate 78: A mixture of phosphonic acid 60 (35 mg, 0.11mmol), L-phenylalanine n-butyl ester hydrochloride (55 mg, 0.21 mmol),phenol (50 mg, 0.53 mmol) and triethylamine (0.2 mL, 1.43 mmol) inpyridine (0.5 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.16 g, 0.74 mmol) andtriphenylphosphine (0.20 g, 0.75 mmol) in pyridine (0.5 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphoamidate (18 mg, 28%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d,1H), 7.25-6.97 (m, 10H), 6.00 (m, 1H), 5.80 (s, broad, 1H), 5.30 (dd,1H), 5.17 (dd, 1H), 4.78 (d, 2H), 4.40-4.03 (m, 7H), 3.85-3.65 (m, 4H),3.45-3.25 (m, 2H), 2.95-2.86 (m, 2H), 1.57 (m, 2H), 1.30 (m, 2H), 0.96(m, 3H); ³¹P NMR (CDCl₃) δ 21.89, 21.09.

Example 72

Monophosphoamidate 79: A mixture of phosphonic acid 60 (0.10 g, 0.30mmol), L-phenylalanine isobutyl ester hydrochloride (0.16 g, 0.61 mmol),phenol (0.14 g, 1.52 mmol) and triethylamine (0.7 mL, 5.02 mmol) inpyridine (2.0 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.12 mmol) andtriphenylphosphine (0.56 g, 2.14 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) to give the monophosphoamidate (19 mg, 10%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (d,1H), 7.25-7.03 (m, 10H), 6.00 (m, 1H), 5.30 (dd, 1H), 5.17 (dd, 1H),4.78 (d, 2H), 4.45 (m, 1H), 4.35-4.18 (m, 4H), 3.95-3.60 (m, 5H), 3.35(m, 1H), 3.00-2.83 (m, 2H), 1.85 (m, 1H), 0.96 (m, 6H); ³¹P NMR (CDCl₃)δ 21.90, 21.07.

Examples 73 to 76 Related to Scheme 22 Example 73

Monophosphoamidate 80: A mixture of monophosphonic acid 6 (0.10 g, 0.30mmol), L-alanine cyclobutyl ester hydrochloride (0.11 g, 0.61 mmol),phenol (0.13 g, 1.4 mmol), and triethylamine (0.51 mL, 3.67 mmol) inpyridine (2 mL) was heated to 60° C. for 5 min. A freshly preparedbright yellow solution of aldrithiol (0.47 g, 2.12 mmol) andtriphenylphosphine (0.56 g, 2.14 mmol) in pyridine (1.0 mL) was added tothe above reaction mixture. The reaction was stirred at 60° C.overnight, cooled to room temperature, and concentrated. The product waspartitioned between EtOAc and saturated NaHCO₃. The organic phase waswashed with brine, dried with Na₂SO₄, filtered, and evaporated underreduced pressure. The crude product was purified by chromatography onISCO (2-propanol/CH₂Cl₂) followed by Gilson HPLC purification(CH₃CN/H₂O) to give the monophosphoamidate (33 mg, 20%, 1:1diastereomeric mixture) as an off-white foam: ¹H NMR (CDCl₃) δ 7.60 (s,1H), 7.30-7.03 (m, 5H), 5.80 (s, broad, 1H), 5.00 (m, 1H), 4.80 (d, 2H),4.28-4.05 (m, 3H), 3.90 (m, 4H), 3.03 (s, broad, 1H), 2.35 (m, 2H), 2.05(m, 2H), 1.80 (m, 2H), 1.30 (m, 3H), 0.90 (m, 2H), 0.62 (m, 2H); ³¹P NMR(CDCl₃) δ 21.91, 20.61.

Example 74

Bisphosphoamidate 81: A mixture of phosphoric acid 6 (60 mg, 0.18 mmol),L-alanine cyclobutyl ester hydrochloride (0.13 g, 0.72 mmol), andtriethylamine (0.31 mL, 2.16 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.28 g, 1.26 mmol) and triphenylphosphine (0.34 g, 1.26mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on silica gel (5% MeOH/CH₂CH₂) togive the bisphosphoamidate (30 mg, 28%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.60 (s, 1H), 5.70 (s, 1H), 5.00 (m, 2H), 4.90 (s, 2H), 4.25(m, 2H), 4.00 (m, 2H), 3.90 (m, 2H), 3.78 (m, 2H), 3.40 (m, 1H), 3.21(m, 1H), 3.03 (s, broad, 1H), 2.35 (m, 4H), 2.05 (m, 4H), 1.90-1.65 (m,4H), 1.32 (m, 6H), 0.90 (m, 2H), 0.60 (m, 2H); ³¹P NMR (CDCl₃) δ 20.70.

Example 75

Bisphosphoamidate 82: A mixture of phosphoric acid 6 (60 mg, 0.18 mmol),L-alanine cyclopentyl ester hydrochloride (0.13 g, 0.72 mmol), andtriethylamine (0.31 mL, 2.16 mmol) in pyridine (1.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.28 g, 1.26 mmol) and triphenylphosphine (0.34 g, 1.26mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on silica gel (5% MeOH/CH₂Cl₂) togive the bisphosphoamidate (30 mg, 27%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.62 (s, 1H), 5.72 (s, 1H), 5.20 (m, 2H), 4.80 (s, 2H), 4.25(m, 2H), 4.04-3.88 (m, 4H), 3.74 (m, 2H), 3.40 (m, 1H), 3.23 (m, 1H),3.03 (s, broad, 1H), 1.95-1.58 (m, 16H), 1.37 (m, 6H), 0.90 (m, 2H),0.60 (m, 2H); ³¹P NMR (CDCl₃) δ 20.64.

Example 76

Bisphosphoamidate 83: A mixture of phosphonic acid 6 (40 mg, 0.12 mmol),L-phenylalanine cyclobutyl ester hydrochloride (0.13 g, 0.48 mmol), andtriethylamine (0.20 mL, 1.44 mmol) in pyridine (0.5 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.19 g, 0.85 mmol) and triphenylphosphine (0.22 g, 0.85mmol) in pyridine (0.5 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on silica gel (5% MeOH/CH₂Cl₂) togive the bisphosphoamidate (20 mg, 22%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.50 (s, 1H), 7.28-7.05 (m, 10H), 5.72 (s, 1H), 5.00 (m, 2H),4.90 (s, 2H), 4.23-4.03 (m, 4H), 3.68 (m, 2H), 3.42-3.19 (m, 2H),3.15-2.82 (m, 7H), 2.38 (m, 4H), 2.00 (m, 4H), 1.85-1.55 (m, 4H), 0.90(m, 2H), 0.60 (m, 2H); ³¹P NMR (CDCl₃) δ 20.31.

Examples 77 and 78 Relate to Scheme 23 Example 77

Diisopropyl Phosphonate 84: A mixture of 4 (5.0 g, 12.82 mmol) andtrifluoroethylamine (6.35 g, 64.10 mmol) in CH₃CN (40 mL) was placed ina reaction bomb and heated to 80° C. for 4 h. The reaction mixture wascooled to room temperature and concentrated under reduced pressure. Theproduct was partitioned between 15% MeOH/CH₂CL (3×) and brine, driedwith Na₂SO₄, filtered, and concentrated. The crude product was purifiedby chromatography on ISCO (2-propanol/CH₂Cl₂) followed by Gilson HPLCpurification (CH₃CN/20) to give 84 (3.26 g, 56%) as a pale yellow foam.

Example 78

Bisphosphoamidate 85: A mixture of phosphonic acid 42 (0.11 g, 0.29mmol), L-alanine cyclobutyl ester hydrochloride (0.31 g, 1.75 mmol), andtriethylamine (0.52 mL, 3.67 mmol) in pyridine (2.0 mL) was heated to60° C. for 5 min. A freshly prepared bright yellow solution ofaldrithiol (0.47 g, 2.12 mmol) and triphenylphosphine (0.56 g, 2.12mmol) in pyridine (1.0 mL) was added to the above reaction mixture. Thereaction was stirred at 60° C. overnight, cooled to room temperature,and concentrated. The product was partitioned between EtOAc andsaturated NaHCO₃. The organic phase was washed with brine, dried withNa₂SO₄, filtered, and evaporated under reduced pressure. The crudeproduct was purified by chromatography on ISCO (2-propanol/CH₂Cl₂) togive the bisphosphoamidate (97 mg, 54%) as a pale yellow foam: ¹H NMR(CDCl₃) δ 7.65 (s, 1H), 5.90 (s, broad, 1H), 5.00 (m, 2H), 4.80 (s, 2H),4.35-4.20 (m, 4H), 4.00 (m, 2H), 3.87 (m, 2H), 3.70 (d, 2H), 3.38 (m,1H), 3.20 (m, 1H), 2.30 (m, 4H), 2.00 (m, 4H), 1.90-1.60 (m, 4H), 1.35(m, 6H); ³¹P NMR (CDCl₃) δ 20.61.

Example 79

This example teaches assays used to demonstrate antiproliferationactivity

Cell Types Used for Anti-Proliferation Assays

Human cancer cell lines used in anti-proliferation assays included sixcervical carcinoma cell lines with three types of HPV (HPV-16, HPV-18,HPV-39), one HPV negative cervical carcinoma cell line, and twokeratinocyte-like carcinoma from tongue. Normal human cells testedincluded skin keratinocytes, cervical keratinocytes, and lungfibroblasts. Skin keratinocytes and cervical keratinocytes were obtainedfrom Cambrex (East Rutherford, N.J.) and all other cells were obtainedfrom American Type Culture Collection (Manassas, Va.). Table 79-1summarizes characteristics of each cell type and culture conditions.Anti-proliferation assay procedure

1. Cell Culture

Cells were detached from culture flasks using trypsin, counted, andplated in 96-well culture plates (250-1000 cells per well, depending oncell type). On the next day (defined as day 0), after cells attached tothe bottom of plates, 5-fold serial dilutions of compounds were added induplicate. No compound and 10 μM colchicine (cell division inhibitor)was added to control wells, which would represent 100% proliferation and0% proliferation, respectively.

2. Staining of Cells with Sulforhodamine B

Seven days after addition of compounds, culture plates were treated with10% trichloroacetic acid at 4° C. for 1 hr, then washed with water. Thisprocedure allows cell-derived proteins to bind to the bottom surface ofplates. Proteins were stained with 0.4% Sulforhodamine B in 1% aceticacid for 10 minutes, followed by extensive washing with 1% acetic acid.Remaining dye bound to the bottom of plates was dissolved in 10 mMTrizma base. This generated purple color that was quantified bymeasuring the absorbance at 510 nm wavelength, using spectrophotometer.

3. Data Analysis

From the experimental data, sigmoidal dose-response curve was generatedand 50% effective concentration (EC₅₀) was calculated using GraphPadPrism version 4.01 for Windows (GraphPad Software, San Diego Calif.USA).

TABLE 79-1 Cell types used in antiproliferation assays Culture Name HPVstatus Origin media* HPV positive carcinoma cell lines SiHa HPV-16Squamous cell carcinoma in cervix A1, A2 (1-2 copies per cell) Ca SkiHPV-16 Epidermoid carcinoma, metastased A1, A2 (600 copies per cell) tosmall intestine from cervix MS751 HPV-18 Epidermoid carcinoma,metastased A1, A2 (also contains a to lymph node from cervix partialHPV-45 genome) HeLa HPV-18 Epithelial adenocarcinoma in cervix A1, A2C-4 I HPV-18 Carcinoma in cervix A1, A2 ME-180 HPV-39 Epidermoidcarcinoma, metastased A1, A2 to omentum from cervix HPV negativecarcinoma cell lines HT-3 None Carcinoma, metastased to lymph A1, A2node from cervix SCC-4 None Squamous cell carcinoma in tongue A1, A2SCC-9 None Squamous cell carcinoma in tongue A1, A2 Cells from normalhuman tissues HEL299 None Fibroblasts in embryonic lung A1, A2 PHK NoneKeratinocytes in adult foreskin B1, B2 (skin keratinocytes) CK (cervicalNone Keratinocytes in adult cervix B1, B2 keratincytes) *Culture mediaCells were maintained in humidified incubators at 37° C. with 5% CO₂, inthe following culture media. A1: Medium for culture maintenance: EagleMEM with Earle's BSS (Cambrex, East Rutherford, NJ), supplemented with10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100μg/mL streptomycin. A2: Medium for antiproliferation assays: Eagle MEMwith Earle's BSS, supplemented with 5% fetal bovine serum, 2 mMglutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. B1:Medium for culture maintenance: Keratinocyte-SFM (Invitrogen, Carlsbad,CA), supplemented with 0.01 mg/mL bovine pituitary extract, 0.001 μg/mLrecombinant epidermal growth factor, 100 units/mL penicillin, and 100μg/mL streptomycin. B2: Medium for antiproliferation assays: 4:1 mixtureof B1 and A2.

Results

1. Selective Antiproliferation Activity of the Amidate Prodrugs in HPVPositive SiHa Cells Compared with Normal Fibroblasts.

The goal was to discover a compound that inhibits growth ofHPV-transformed lesion without affecting normal cells in epidermis anddermis (such as keratinocytes and fibroblasts). In vitroantiproliferation assays were setup using SiHa cells and HEL cells,which model HPV-transformed lesion and normal fibroblasts, respectively.SiHa cells are derived from squamous cell carcinoma in cervix caused byHPV-16 infection and HEL fibroblasts are derived from normal humanembryonic lung (Table 79-1). As shown in Table 79-2, 50% effectiveconcentration (EC₅₀) of the seven amidate prodrugs in SiHa cells ranged0.13-3.2 in, while EC₅₀ in HEL cells ranged 12-727 nM, indicating thatthese compounds inhibited proliferation of SiHa cells more efficientlythan HEL cells. HEL/SiHa selectivity index (HEL EC₅₀ divided by SiHaEC₅₀) ranged from 72-559 (Table 79-2).

All seven amidate prodrugs produce the same metabolite, cprPMEDAP.cprPMEDAP is further metabolized to PMEG [Compton et al., 1999; Haste etal., 1999]. Antiproliferation EC₅₀ of these compounds in SiHa cells weremuch higher than those of the prodrugs (Table 79-2), indicating thatattachment of amidate moieties improved potency. Furthermore, HEL/SiHaselectivity indices of cprPMEDAP and PMEG were 17 and 4.1, respectively(Table 79-2), indicating that the prodrugs have better selectivity thancprPMEDAP and cprPMEDAP has better selectivity than PMEG.

PMEG is known to be phosphorylated to PMEGpp that acts as achain-terminating inhibitor of cellular DNA polymerase [Compton et al.,1999; Haste et al., 1999]. Four known DNA polymerase inhibitors(Cidofovir, Ara C, doxifluridine, and Aphidicolin) and other anticancerdrugs with different mechanisms of action, including DNA topoisomaraseinhibitors (Dacarbazine, Ellipticine), DNA alkylaters (Doxorubicin,Mitoxantrone, Bleomycin, Mechlorethanmine), and tublin inhibitors(Vincristine, Vinblastine, Etoposide, and Indanocine) were tested inSiHa and HEL cells (Table 79-2). Antiproliferation EC₅₀ of thesecompounds in SiHa cells varied, and some were equally or more potentthan the seven amidate prodrugs. Nonetheless, all of them exhibited poorHEL/SiHa selectivity indices (0.01-3.98), compared with the sevenamidate prodrugs.

Taken together, a unique set of compounds were taught, which showssub-low nM antiproliferation EC₅₀ in HPV-16 positive SiHa carcinomacells and greater than 50 fold selectivity when compared with HELfibroblasts.

2. Selective Antiproliferation Activity of the Amidate Prodrugs in HPVPositive SiHa Cells Compared with Normal Keratinocytes

In order to test effect of the compounds in normal cells from epidermis,anti-proliferation assays were performed using primary humankeratinocytes, isolated from skin (PHK) and cervix (CK).Antiproliferation EC₅₀ values obtained with the seven prodrugs in PHKand CK were lower than those in HEL, indicating that keratinocytes aremore susceptible than fibroblasts (Table 79-2 and 79-3). Nonetheless,PHK/SiHa and CK/SiHa selectivity indices of these prodrugs and cprPMEDAPwere still better than the control compounds PMEG and a DNA polymeraseinhibitor AraC (Table 79-3). Thus, the prodrugs preferentially inhibitedproliferation of HPV-16 positive SiHa cells, compared with normalkeratinocytes from skin and cervix.

3. Antiproliferation Activities in other HPV Positive Cells

The seven prodrugs were then tested in five additional cell linesderived from HPV-induced cervical carcinoma (listed in Table 79-1) inantiproliferation assays and data are shown in Table 4 along with SiHadata. In SiHa, C-41, and MS751 cells, all compounds except Compound Cshowed sub-low nM antiproliferation EC₅₀. In CaSki, HeLa, and ME-180,however, all compounds were significantly less potent, with EC₅₀ ranging7.8-410 nM. There seems to be no correlation between resistance and HPVtype (16, 18 or 39), or resistance and metastatis (CaSki, MS751, andME180 are derived from metastased site). The control compound AraC (DNApolymerase inhibitor) uniformly inhibited all cell lines with EC₅₀values ranging 94-257 mM.

4. Antiproliferation Activities in HPV Negative Carcinoma Cells

To investigate the effect of the compounds on HPV negative carcinomacell lines, three cell lines (HT-3, SCC4, SCC9, Table 79-1) were testedin antiproliferation assays. As shown in Table 79-4, all seven prodrugswere equally or more potent than the control compound AraC.

TABLE 79-2 Selective inhibition of HPV16+ SiHa cells compared with HELfibroblasts Antiproliferation EC50 (nM) SiHa cervical Selectivitycarcinoma HEL lung Compound ID. Note (HEL/SiHa) (HPV16) fibroblast A 720.6 43 B 559 1.3 727 C 115 0.20 23 D 135 3.2 431 E 164 0.50 82 F 210 2.5526 G 92 0.13 12 Controls cprPMEDAP Metabolite 17 284 4821 PMEGMetabolite 4.1 207 861 AraC DNA pol inh 0.113 257 29 Cidofovir DNA polinh 0.3 84013 27952 Doxifluridine DNA pol inh 0.449 8755 3927Aphidicolin (+) DNA pol inh 0.40 856 324 Dacarba-zine DNA topo inh 3.987402 29481 Ellipticine DNA topo inh 1.02 478 486 Doxorubicin DNAalkylater 0.43 9.76 4.20 Mitoxantrone DNA alkylater <0.37 8.67 <3.2Mechlorethamine hydrochloride DNA alkylater 1.02 21863 22203 BleomycinDNA alkylater 0.01 3138 20.28 Vincristine Tublin inh 1.55 1.24 1.92Vinblastine Tublin inh 0.39 0.68 0.27 Etoposide Tublin inh 0.31 469 144Indanosine Tublin inh 0.27 588 159

TABLE 79-3 Selective inhibition of HPV16+ SiHa cells compared withprimary keratinocytes Antiproliferation EC50 (nM) SiHa cervicalSelectivity Selectivity carcinoma PHK skin CK cervical Note (PHK/SiHa)(CK/SiHa) (HPV16) keratinocytes keratinocytes Comp ID. A 58 11 0.6 35 7B 75 42 1.3 98 54 C 4 7 0.20 0.8 1.4 D 12 7 3.2 39 22 E 10 11 0.50 5.25.4 F 31 3 2.5 78 7.1 G 22 15 0.13 2.9 1.9 Controls cprPMEDAP metabolite13 2.4 284 3698 694 PMEG metabolite 0.48 2.4 207 101 501 AraC DNA polinh 0.57 0.4 257 147 107

TABLE 79-4 Antiproliferation activities in other HPV positive andnegative carcinoma cells Antiproliferation Antiproliferation EC50 (nM)in EC50 (nM) in HPV HPV positive carcinoma cells negative carcinomacells SiHa Caski HeLa MS-751 C-4I ME-180 HT-3 SCC-4 SCC-9 HPV16 HPV16HPV18 HPV18 HPV18 HPV39 cervix tongue tongue Comp ID. A 0.6 29 16 1.76.5 27 14 17 40 B 1.3 246 410 18 27 254 104 53 150 C 0.20 3.87 6.6 0.541.0 7.8 9.5 2.1 2.5 D 3.2 301 398 16 24 288 127 44 147 E 0.50 38 19 2.403.1 27 17 8 13 F 2.5 124 127 4.2 6.0 41 24 10 28 G 0.13 28 12 0.9 3.18.2 6.0 2.1 7.9 Controls AraC 257 94 174 144 123 101 214 74 68

Example 80 Antiproliferation Assay

Antiproliferation assays measure effect of compounds on proliferation ofcultured cells. Active compounds in antiproliferation assays may becytostatic (inhibit cell division) and/or cytocidal (kill cells). Byperforming antiproliferation assays using HPV positive carcinoma cellsand normal cells, we identify compounds that selectively inhibitproliferation of HPV positive carcinoma cells compared with cells fromnormal human tissues. Table 80-1 summarizes characteristics of each celltype, including six cervical carcinoma cell lines transformed by HPV,normal human skin keratinocytes (PHK), and normal lung fibroblasts(HEL). Skin keratinocytes were obtained from Cambrex (East Rutherford,N.J.). All other cells were obtained from American Type CultureCollection (Manassas, Va.).

Cells were detached from culture flasks using trypsin, counted, andplated in 96-well culture plates (250-100 cells per well, depending oncell type). On the next day (defined as day 0), 5-fold serial dilutionsof compounds were added in duplicate. Seven days after addition ofcompounds, culture plates were treated with 10% trichloroacetic acid at4° C. for 1 hr and washed with water. This procedure allows cellularproteins to bind to the bottom surface of plates. Proteins were stainedwith 0.4% Sulforhodamine B in 1% acetic acid for 10 minutes, followed byextensive washing with 1% acetic acid. Remaining dye bound to the bottomof plates was solubilized in 10 mM Trizma base, generating purple color.Intensity of the color (proportional to cell number) was quantified bymeasuring the absorbance at 510 nm wavelength, using spectrophotometer.Cells without drug treatment (=100% proliferation) and cells treatedwith 10 μM colchicine (cell division inhibitor) (=0% proliferation) wereused as controls, to determine % inhibition. % inhibition values wereplotted against compound concentrations, fitted to a sigmoidal doseresponse curve, from which the compound concentration that reduced cellproliferation rate by 50% (=EC₅₀)) was determined. GraphPad Prismversion 4.00 for Windows (GraphPad Software, San Diego Calif. USA) wasused for the curve fitting and EC₄₀ calculation.

Apoptosis Assay (Caspase 3 Induction Method)

Induction of caspases is one of the early events associated withapoptosis or programmed cell death. Caspase activity can bequantitatively detected using fluorescent substrate. Compounds thatdirectly act on the apoptotic pathway may induce caspase in a relativelyshort incubation period (<24 hrs). Compounds that disturb other cellphysiology, which eventually causes apoptosis, may require longerincubation period (>48 hrs) for induction of caspase.

10,000 cells were plated in 96-well culture plates and incubated with5-fold serial dilutions of compounds for 24, 48, and 72 hrs. Cells werelysed and activity of caspase in cell lysates were measured usingfluorescent substrate, according to the manufacturer's instruction(Caspases assay kit, Roche, Indianapolis, Ind.).

Apoptosis Assay (Annexin V Staining Method)

Translocation of phosphatidylserine from the inner of the cell membraneto the outside is one of the early/intermediate events associated withapoptosis or programmed cell death. Translocated phosphatidylserine canbe detected by incubating cells with FITC-labelled Annexin V, which is aCa++ dependent phospholipid-binding protein. When cells are stained withAnnexin-FITC and propidium iodide (which stains dead cells), live cellsare negative for both dyes, dead cells are positive for both, whileapoptotic cells are positive only for Annexin-FITC.

HPV-16 SiHa cells were cultured with three different concentrations ofcompounds for 3 or 7 days and simultaneously stained with Annexin-FITCand propidium iodide. Staining of each individual cell was examined byflow cytometry.

Results Selective Antiproliferation Activity

The purpose of this procedure was to identify compounds that inhibitsgrowth of HPV-transformed lesion without affecting normal cells inepidermis and dermis (such as keratinocytes and fibroblasts). Therefore,compounds were tested in SiHa, PHK, and HEL cells, which modelHPV-transformed cells, normal keratinocytes, and normal fibroblasts,respectively.

Representative compounds of the present invention, such as those listedin Table 80-2, showed detectable levels of antiproliferation activity inSiHa cells, with 50% effective concentration (EC₅₀) less than 25,000 nM.Active compounds were also tested in HEL cells. In all cases, EC₅₀ inHEL cells were higher than EC₅₀ in SiHa cells, indicating that theactive compounds inhibited proliferation of SiHa cells more efficientlythan HEL cells. Other nucleotide/nucleoside analogs, such as PMEG(2-phosphonomethoxyethyl guanine), Ara-C (cytarabine, CAS#147-94-4), andgemcitabine (CAS#95058-81-4) did not show such selectivity. Podofilox(CAS#518-28-5), the active ingredient of the anti-wart drug Condylox,also showed no selectivity.

Representative prodrug compounds of the present invention, such as thoselisted in Table 80-3 show activities. In most cases, the prodrugs weremore potent and in some cases, more selective than their respectiveparent compounds. The majority of phosphoamidate prodrugs were moreactive and selective than podofilox.

Taken together, compounds were identified that possess sub nMantiproliferation EC₅₀ in HPV-16 positive SiHa cells and greater than 50fold selectivity when compared with PHK keratinocytes or with HELfibroblasts.

Antiproliferation Activity in Other HPV+ Cell Lines

Selected compounds were also tested in five additional cell linesderived from HPV-induced cervical carcinoma (see Example 79 and Table80-4). Each compound showed different levels of activities in the sixHPV+ cell lines, regardless the type of HPV present. In general,compounds were more potent in SiHa (HPV-16), C-41 (HPV-18), and MS751(HPV-18) cells than in CaSki (HPV-16), HeLa (HPV-18), and ME-180(HPV-39) cells.

Induction of Apoptosis (Caspase 3 Induction Method)

A representative compound of the present invention was tested forinduction of apoptosis in SiHa cells. When cells were incubated for 72hrs (solid bars), significant dose responsive induction of caspase wasobserved, indicating that the compound induced apoptosis (FIG. 80-1).Induction of caspase was less obvious with 48 hr incubation (shadedbars) and was not observed with 24 hr incubation (data not shown).

Induction of Apoptosis (Annexin V Staining Method)

PMEG, N6-cyclopropyl PMEDAP, and a representative compound of thepresent invention were tested at three different concentrations, forinduction of apoptosis in SiHa cells, using Annexin V-Propidium iodidedouble staining method. With all three compounds, a greater percentageof apoptotic cells were observed on day 7 than day 3. The aforementionedrepresentative compound of the present invention was the most active ininducing apoptosis; on day 7, 63.8% of cells in the culture treated with0.2 μg/m of this compound were apoptotic. In contrast, cultures treatedwith 0.2 μg/mL PMEG and 0.5 μg/mL N6-cyclopropyl PMEDAP only had 1.2%and 15.9% of apoptotic cells, respectively.

TABLE 80-1 Cell types used in antiproliferation assays Culture Name HPVstatus* Origin media** HPV positive carcinoma cell lines SiHa HPV-16Squamous cell carcinoma in cervix A1, A2 Ca Ski HPV-16 Epidermoidcarcinoma, metastased A1, A2 to small intestine from cervix MS751 HPV-18Epidermoid carcinoma, metastased A1, A2 (also to lymph node from cervixcontains a partial HPV- 45 genome) HeLa HPV-18 Epithelial adenocarcinomain cervix A1, A2 C-4 I HPV-18 Carcinoma in cervix A1, A2 ME-180 HPV-39Epidermoid carcinoma, metastased A1, A2 to omentum from cervix Cellsfrom normal human tissues HEL299 none Fibroblasts in embryonic lung A1,A2 PHK none Keratinocytes in adult foreskin B1, B2 (skin keratinocytes)*The subtype of HPV DNA integrated in the cellular DNA. **Culture mediaCells were maintained in humidified incubators at 37° C. with 5% CO₂, inthe following culture media. A1: Medium for culture maintenance: EagleMEM with Earle's BSS (Cambrex, East Rutherford, NJ), supplemented with10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100μg/mL streptomycin. A2: Medium for antiproliferation assays: Eagle MEMwith Earle's BSS, supplemented with 5% fetal bovine serum, 2 mMglutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. B1:Medium for culture maintenance: Keratinocyte-SFM (Invitrogen, Carlsbad,CA), supplemented with 0.01 mg/mL bovine pituitary extract, 0.001 μg/mLrecombinant epidermal growth factor, 100 units/mL penicillin, and 100μg/mL streptomycin. B2: Medium for antiproliferation assays: 4:1 mixtureof B1 and A2.

TABLE 80-2 Antiproliferation activity of N6-substituted PMEDAP in HPV16+SiHa cells and HEL fibroblasts

where R^(X1) is hydrogen and R^(X2) is one of the followingsubstituents, except in the indicated instances (*), where R^(X1) andR^(X2) together forms a N-heterocyclic ring. methylamine 1-propylamine1-butylamine dimethylamine methylethylamine 2-methylpropan-1-amine2-propynyl amine 2-butyleneamine 2-isobutylen amine cyclopropylaminecyclopropylmethanamine 1-cyclopropylethanamine dicyclopropylaminecyclobutylamine cyclopentanamine cyclohexaneamine cycloheptanaminecyclooctanamine diethanolamine 2-ethanolamine 2-propanol amine 1-amino2-propanol amine 2-methoxyethylamine 6-aminohexaneamine3-aminopropylamine 2-dimethylaminoethylamine 6-hexanateamine benzylaminemethylbenzylamine 4-aminobenzylamine 2-phenylethanamine 2-pyridinyl1-methanamine 3-pyridinyl 1-methanamine 4-pyridinyl 1-methanamine1-naphthylamine *pyrrolidine (N6 makes pyrrolidine) *piperidine (N6makes piperidine) *morpholine (N6 makes morpholine)2,2,2-trifluoroethanamine

TABLE 80-3 Antiproliferation activity of phosphoamidate prodrugs of N6-substituted PMEDAP in HPV16+ SiHa cells, PHK keralinocytes, and in HELfibroblasts

where R^(X1) and R^(X2) are substituted as depicted in the formula, andY^(1A) and Y^(1B) are substituted as indicated, Y^(1A) Y^(1B) OH OH POCPOC O-iPr O-iPr Ala-Et Ala-Et Ala-Pr Ala-Pr Ala-iPr Ala-iPr Ala-BuAla-Bu Ala-cBu Ala-cBu Ala-cPen Ala-cPentyl Ala-Hexyl Ala-HexylAla-Octyl Ala-Octyl Aba-Et Aba-Et Aba-Bu Aba-Bu Aba-Octyl Aba-OctylPhe-Et Phe-Et Phe-Bu Phe-Bu Phe-iBu Phe-iBu Phe-cBu Phe-cBu OPh Ala-MeOPh Ala-Et OPh Ala-Pr OPh Ala-iPr OPh Ala-Bu OPh Ala-tBu OPh Ala-HexylOPh Ala-Octyl OPh Aba-Et OPh Aba-Bu OPh Aba-cBu OPh Aba-Octyl OPh Phe-EtOPh Phe-Bu OPh Phe-iBu OPh D-Ala-Me

where R^(X1) and R^(X2) are substituted as depicted in the formula, andY^(1A) and Y^(1B) are substituted as indicated, Y^(1A) Y^(1B) OH OHO-iPr O-iPr POC POC Ala-Et Ala-Et Ala-Bu Ala-Bu Ala-cBu Ala-cBuAla-Hexyl Ala-Hexyl Aba-Bu Aba-Bu Phe-Et Phe-Et Phe-Bu Phe-Bu Phe-iBuPhe-iBu OPh Ala-Et OPh Ala-Bu OPh Ala-cBu OPh Ala-Hexyl OPh Aba-Bu OPhPhe-Et OPh Phe-Bu OPh Phe-iBu

where R^(X1) and R^(X2) are substituted as depicted in the formula, andY^(1A) and Y^(1B) are substituted as indicated, Y^(1A) Y^(1B) OH OHO-iPr O-iPr Ala-Bu Ala-Bu OPh Phe-Et

where R^(X1) and R^(X2) are substituted as depicted in the formula, andY^(1A) and Y^(1B) are substituted as indicated, Y^(1A) Y^(1B) OH OHAla-Bu Ala-Bu

TABLE 80-4 Antiproliferation activities of N6-cycloprolyl PMEDAP and itsphosphoamidate prodrugs in six different HPV positive cellsAntiproliferation EC50 (nM) in HPV positive carcinoma cells SiHa CaSkiHeLa MS-751 C-4I ME-180 Compound ID. HPV16 HPV16 HPV18 HPV18 HPV18 HPV39A 0.6 29 16 1.7 6.5 27 B 1.3 246 410 18 27 254 C 0.2 3.9 6.6 0.5 1.0 7.8D 3.2 301 398 16 24 288 E 0.5 38 19 2.4 3.1 27 F 2.5 124 127 4.2 6.0 41G 0.13 28 12 0.9 3.1 8.2 H 0.03 2.0 0.7 0.04 0.44 1.8 (cprPMEDAP) 28414149 6926 3313 1332 8315

Example 81 Rabbit Skin Irritation Study of Compounds A and B

A study was conducted to evaluate the potential of two compounds of thepresent invention to produce irritation when administered via dermalapplication to male rabbits for seven consecutive days. A total of sixmales were assigned to the study as presented in the table below.

Group Assignments Number of Animals Group Number Test Article^(a) (male)1 Compound B^(b) 3 2 Compound A^(c) 3 ^(a)Each animal received dermaltreatments of vehicle (placebo gel), one positive control article, andthree concentrations of the appropriate test article. Each animalreceived one test article at concentrations of 0.01, 0.03, and 0.1%.^(b)The positive control article used was 0.1%9-(2-phosphonylmethoxyethyl) guanine (PMEG). ^(c)The positive controlarticle used was 1% Cidofovir ®.

The vehicle, positive control articles, and test articles wereadministered dermally once daily for seven days during the study. Thetest articles were administered at concentrations of 0.01, 0.03, and0.1%. The positive control articles were administered at concentrationsof 0.1% (PMEG) or 1% (Cidofovir®). The dose volume for all formulationswas a fixed volume of 100 μL.

The test sites for each animal were shaved prior to the initialadministration and as needed during the study. Two sites were clipped onthe left dorsal side, and three were clipped on the right dorsal side.The outline of each dosing (approximately 1 square inch each) was markedwith indelible ink. The total clipped area comprised no less than 10% ofthe total body surface of each animal. The vehicle and appropriatepositive control and test article were administered to each animalwithin a dosing site of approximately 1 square inch. Vehicle wasadministered on the left rostral site (Dose Site 1), and the appropriatepositive control article was administered to the left caudal site (DoseSite 2). The appropriate test article was administered as follows: 0.01%to the right rostral site (Dose Site 3), 0.03% to the right middle site(Dose Site 4), and 0.1% to the right caudal site (Dose Site 5). Collarswere placed on the animals immediately following dosing for 1 to 2hours.

The sites were evaluated for erythema and edema prior to dosing on Day 1and daily thereafter, approximately 24 hours following each dose andprior to the next dose. Each site was assigned an irritation score basedupon the Draize scale for scoring skin irritation (Draize J H, WoodardG, Calvery H O, Methods for the study of irritation and toxicity ofsubstances applied topically to the skin and mucous membranes. JPharmacol Exp Ther 1944; 82:377-90).

Observations for mortality, morbidity, and the availability of food andwater were conducted twice daily for all animals. Detailed clinicalexaminations were conducted prior to randomization, prior to dosing onDay 1, and daily thereafter. Body weights were measured and recorded theday after arrival, prior to randomization, and prior to dosing on Days1, 3, and 7.

Euthanasia was by intravenous anesthesia overdose with sodiumpentobarbital-based euthanasia solution and exsanguinations by severingthe femoral vessels. The animals were examined carefully for externalabnormalities including masses. The skin was reflected from a ventralmidline incision and any abnormalities were identified and correlatedwith ante-mortem findings. The abdominal, thoracic, and cranial cavitieswere examined for abnormalities and the organs removed, examined, and,where required, placed in neutral buffered formalin. The dosing sites,kidneys, and any gross lesions of each animal were collected andpreserved. Microscopic examination of fixed hematoxylin andeosin-stained paraffin sections were performed for each dosing site forall animals. The slides were examined by a veterinary pathologist. Afour-step grading system was utilized to define gradable lesions forcomparison between dose groups.

Conclusions

The two test articles did not produce notable clinical findings, dermalirritation, changes in body weight or macroscopic and microscopicobservations at any dose concentrations. One of the positive controlswas associated with clinical findings and slight to moderate macroscopicand microscopic observations.

Example 82 Rabbit Skin Irritation Study of Compounds B and H

A study was conducted to evaluate the potential of two compounds of thepresent invention to produce irritation when administered via dermalapplication to male rabbits for seven consecutive days. A total of 24males were assigned to the study.

Study Design for Compound B Group 1 Group 2 Group 1 Concentration^(b)Group 2 Concentration^(b) Dose Site (n = 6)^(a) % (mg/mL) (n = 6)^(a) %(mg/mL) 1 Vehicle 0.0 0.0 PMEG 0.1% 1.0 control (positive control) 2 LowDose 0.03 0.3 Low Dose 0.03 0.3 3 Mid Dose 0.1 1.0 Mid Dose 0.1 1.0 4High Dose 0.3 3.0 High Dose 0.3 3.0 ^(a)Each group consisted of sixnaïve rabbits. ^(b)The vehicle for Dose Site 1, Group 1, and Dose Site1, Group 2 (PMEG) was the vehicle gel. The vehicle for Group 1 treatedsites was the vehicle gel, and the vehicle for Group 2 treated sites wasthe vehicle ointment.

Study Design for Compound H Group 3 Group 4 Group 3 Concentration^(b)Group 4 Concentration^(b) Dose Site (n = 6)^(a) % (mg/mL) (n = 6)^(a) %(mg/mL) 1 Vehicle 0.0 0.0 PMEG 1.0% 10.0 control (positive control) 2Low Dose 0.03 0.3 Low Dose 0.03 0.3 3 Mid Dose 0.1 1.0 Mid Dose 0.1 1.04 High Dose 0.3 3.0 High Dose 0.3 3.0 ^(a)Each group consisted of sixnaïve rabbits. ^(b)The vehicle for Dose Site 1, Group 3 was the vehicleointment, and the vehicle for Dose Site 1, Group 4 (cPrPMEDAP) was thevehicle gel. The vehicle for Group 3 treated sites was the vehicle gel,and vehicle for Group 4 treated sites was the vehicle ointment.

The test and control articles were administered dermally once per dayfor 7 consecutive days during the study. The dose levels for Compound Bwere 0.03, 0.1, and 0.3%. The dose levels for Compound H were 0.03, 0.1,and 0.3%. The dose level for PMEG (positive control) was 0.1%. The doselevel for cPrPMEDAP (positive control) was 1.0%. The dose level for thevehicle control was 0.0% (this was dosed as both gel and ointmentformulations). The dose volume for all sites was a constant 100 μL. Lessthan 24 hours prior to the first administration, the hair was clippedfrom the back of the animal. This clipped area comprised no less than10% of the total body surface area. Care was taken to avoid abrading theskin.

The test, positive control, and vehicle control articles wereadministered within a dosing site of approximately 1″×1″. Two dosingsites were placed along the left dorsal surface. The vehicle or positivecontrol article was administered to the rostral site, and the low doseof the test article was administered to the caudal site. Two dosingsites were placed along the right dorsal surface. The mid-dose of thetest article was administered to the rostral site, and the high dose ofthe test article was administered to the caudal site. Collars wereplaced on the animals for approximately two hours immediately followingdosing. The duration of collaring was documented in the raw data.

Observations for mortality, morbidity, and the availability of food andwater were conducted twice daily for all animals. The test sites wereevaluated for erythema and edema prior to the first administration andat approximately 24 hours following each administration (prior to thenext scheduled dosing) and daily during the 7-day recovery period.Observations for clinical signs were conducted daily during the study atthe same time as the dermal observations. Body weights were measured andrecorded the day after receipt, prior to randomization, prior to testarticle administration on Day 1, and on Days 7 and 14, and at necropsy(Days 8 and 15). Body weights taken at receipt and prior to random arenot reported, but maintained in the study file. Blood samples (4-6 mL)will be collected from 6 animals/group at termination and 3animals/group at recovery from the jugular or other suitable vein forevaluation of clinical pathology parameters.

Additional blood samples (approximately 1 mL) were taken from allanimals from the jugular or other suitable vein for determination of theplasma concentrations of the test article at approximately 2 hourspostdose on Day 7. Samples were placed in tubes containing potassiumEDTA and stored on an ice block until centrifuged. Animals were notfasted before blood collection. Samples were stored at −70° untilexamination.

Complete necropsy examinations were performed under procedures approvedby a veterinary pathologist on all animals. Euthanasia was by anesthesiaoverdose with sodium pentobarbital-based euthanasia solution via the earvein/artery or other suitable vein and exsanguinations by severing thefemoral vessels. The animals were examined carefully for externalabnormalities including masses. The skin was reflected from a ventralmidline incision and any abnormalities were identified and correlatedwith antemortem findings. The abdominal, thoracic, and cranial cavitieswere examined for abnormalities and the organs removed, examined, and,where required, placed in neutral buffered formalin. Microscopicexamination of fixed hematoxylin and eosin-stained paraffin sections wasperformed on sections of tissues from the dosing sites (4 per animal),kidneys, and any gross lesions.

At the time of necropsy, Day 8 for main study animals and Day 15 forrecovery animals, the four dosing sites per animal were identified.Approximately half of each dosing site was excised and then collectedand preserved as mentioned above for histologic processing. While theother approximate half of each dosing site was still intact on theanimal, the following procedures are performed. The dosing sites werewiped with three gauzes of ethanol (95%) and allowed to dry completely.Tape (3M® packing tape or equivalent) was applied to each dosing siteten times. A clean piece of tape was used for reach application. Theremaining portions of the dosing sites were then excised with scissors.The scissors were washed between each dose site and animal with acetoneor ethanol. The order of dose site removal was vehicle or positivecontrol site, low dose site, mid-dose site, and high dose site. A 1 cm²tissue was excised from each dose site. The tissue sample was weightedand recorded. The skin punches were minced with clean scissors inindividual appropriately sized scintillation vials. Coldphosphate-buffered saline (5 mL) was added to the scintillation vial.The tissue was then homogenized with 20 second pulses using a mechanicalhomogenizer. The homogenates were quickly frozen at approximately −20°C.

Conclusions

Based on dermal irritation scores and microscopic findings, one of thetest articles was nod-irritating in the vehicle gel, but was a mild tomoderate irritant in the vehicle ointment. The second test article was avery slight irritant in the vehicle gel and a mild irritant whenformulated in the vehicle ointment.

Example 83 Preparation of Topical Gel Pharmaceutical Composition

This example illustrates the preparation of a representative topical gelcomposition containing an active compound of Formula I.

A topical gel composition is prepared having the following composition:

Components % w/w Active compound X* pH 4.5 or 7 Buffer 25 PropyleneGlycol, USP 25 Hydroxyethylcellulose, NF 1.25 Propylparaben, NF 0.01Methylparaben, NF 0.09 Edetate Disodium, USP 0.025 Glycerin, USP 10.00Citric Acid, USP 0.50 Sterile Water for Injection, USP 38.125 *X =Compound ranging from 0.01% to 1.0%

Other compounds of Formula I, such as those prepared in accordance withthe present Specification can be used as the active compound in thepreparation of the gel formulations of this example.

The following ingredients have also been evaluated for suitabilityduring the development of this formulation:

-   -   Isopropyl mysritate (solvent/cosolvent/penetration enhancer),    -   Polyethylene glycols, Triacetin (solvents),    -   Cetyl alcohol and Stearyl alcohol (Stiffening agents),    -   Carbomer (Viscosity enhancer), and    -   Tweens, Spans (emulsifiers).

Example 84 Preparation of Topical Ointment Pharmaceutical Composition

This example illustrates the preparation of a representative topicalointment composition containing an active compound of Formula I.

A topical ointment composition is prepared having the followingcomposition:

Components % w/w Active Compound X* Petrolatum, USP 94.0  SorbitanSesquioleate, NF 0.5 Propylene Glycol, USP 4.5 *X = Compound rangingfrom 0.01% to 1.0%

Other compounds of Formula I, such as those prepared in accordance withthe present Specification can be used as the active compound in thepreparation of the ointment formulations of this example.

The following ingredients have also been evaluated for suitabilityduring the development of this formulation:

-   -   Isopropyl mysritate (solvent/cosolvent/penetration enhancer),    -   Polyethylene glycols, Triacetin (solvents),    -   Cetyl alcohol and Stearyl alcohol (Stiffening agents),    -   Carbomer (Viscosity enhancer), and    -   Tweens, Spans (emulsifiers).

1. A compound of the following structure:


2. A pharmaceutical composition comprising an effective amount of acompound according to claim 1, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable carriers.
 3. Thepharmaceutical composition of claim 2, further comprising one or two ormore therapeutically active agents selected from antiviral agents,antibiotics, antipyretics, or analgesics.
 4. The pharmaceuticalcomposition of claim 2, further comprising one or two or moretherapeutically active agents selected from amantidine, rimantadine andribavirin.